Antisense modulation of hydroxysteroid 11-beta dehydrogenase 1 expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of hydroxysteroid 11-beta dehydrogenase 1. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding hydroxysteroid 11-beta dehydrogenase 1. Methods of using these compounds for modulation of hydroxysteroid 11-beta dehydrogenase 1 expression and for treatment of diseases associated with expression of hydroxysteroid 11-beta dehydrogenase 1 are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods formodulating the expression of hydroxysteroid 11-beta dehydrogenase 1. Inparticular, this invention relates to compounds, particularlyoligonucleotides, specifically hybridizable with nucleic acids encodinghydroxysteroid 11-beta dehydrogenase 1. Such compounds have been shownto modulate the expression of hydroxysteroid 11-beta dehydrogenase 1.

BACKGROUND OF THE INVENTION

[0002] The adrenal gland produces three kinds of steroid hormones:glucocorticoids, mineralocorticoids and adrenal androgens. Cortisol andcorticosterone are the essential human glucocorticoids which playimportant roles in salt and water homeostasis and blood pressurecontrol. Clinical conditions arising from an excess or deficiency ofglucocorticoids or mineralocorticoids include Cushing's syndrome,primary aldosteronism, Addison's disease and congenital adrenalhyperplasia (Shimojo and Stewart, J. Endocrinol. Invest., 1995, 18,518-532).

[0003] Obesity is associated with adverse metabolic consequences such asdiabetes and dyslipidemia. One identified cause of viceral obesity andmetabolic complications is exposure to excessive levels ofglucocorticoids (Bjorntorp and Rosmond, Nutrition, 2000, 16, 924-936).

[0004] The enzyme hydroxysteroid 11-beta dehydrogenase 1 (also known asHSD11B1, HSD-11 and 11-beta-OHSD) plays a pivotal role in determiningintracellular glucocorticoid concentrations by regenerating activeglucocorticoid (cortisol in humans, corticosterone in rats and mice)from inactive cortisone and 11-dehydrocorticosterone and has beensuggested to serve as a tissue-specific amplifier of gucocorticoidaction (Bjorntorp and Rosmond, Nutrition, 2000, 16, 924-936).

[0005] Hydroxysteroid 11-beta dehydrogenase 1 has been cloned and mappedto chromosome 1q32-q34 (Tannin et al., J. Biol. Chem., 1991, 266,16653-16658). It is widely expressed, most notably in liver, lung,adipose tissue, vasculature, ovary and the central nervous system (Seckland Walker, Endocrinology, 2001, 142, 1371-1376).

[0006] Rask et al. have recently demonstrated elevated levels ofhydroxysteroid 11-beta dehydrogenase 1 activity in adipose tissue fromobese humans and impaired activity in the liver (Rask et al., J. Clin.Endocrinol. Metab., 2001, 86, 1418-1421).

[0007] Masuzaki et al. have created transgenic mice which overexpresshydroxysteroid 11-beta dehydrogenase 1 selectively in adipose tissue toan extent similar to that found in adipose tissue of obese humans. Thesemice had increased adipose levels of corticosterone and developedvisceral obesity that was exaggerated by a high-fat diet. The mice alsoexhibited pronounced insulin-resistant diabetes and hyperlipidemia,leading to a proposal that increased adipocyte hydroxysteroid 11-betadehydrogenase 1 activity may be a common molecular etiology for visceralobesity (Masuzaki et al., Science, 2001, 294, 2166-2170).

[0008] Mice homozygous for a targeted deletion of hydroxysteroid 11-betadehydrogenase 1 gene are viable and developmentally normal but cannotregenerate active corticosterone from inert 11-dehydrocorticosterone invivo, demonstrating that hydroxysteroid 11-beta dehydrogenase 1 is thesole 11-beta-reductase in the body (Kotelevtsev et al., Proc. Natl.Acad. Sci. U.S.A., 1997, 94, 14924-14929). The hydroxysteroid 11-betadehydrogenase 1-deficient mice show attenuated activation ofglucocorticoid-sensitive hepatic gluconeogenic enzymes in response tostress or high-fat diets and have a disease resistant phenotype(Kotelevtsev et al., Proc. Natl. Acad. Sci. U.S.A., 1997, 94,14924-14929).

[0009] The thiazolidinedione (TZD) class of antidiabetic agents that areligands for peroxisome proliferator-activated receptor-gamma have beenshown to reduce adipocyte hydroxysteroid 11-beta dehydrogenase 1 mRNAand activity both in vivo and in vitro. Because TZDs preferentiallyreduce visceral fat accumulation in humans, suppression of adiposehydroxysteroid 11-beta dehydrogenase 1 by TZDs could be a mechanism forthis fat redistribution and may play a role in their antidiabeticeffects (Berger et al., J. Biol. Chem., 2001, 276, 12629-12635).

[0010] These findings strongly suggest that increased adipocytehydroxysteroid 11-beta dehydrogenase 1 is a common molecular mechanismfor visceral obesity and diabetes and may be an appropriatepharmaceutical target for the treatment of these disorders.

[0011] In addition to the TZD class of antidiabetic agents discussedabove, other known small molecule inhibitors of hydroxysteroid 11-betadehydrogenase 1 include glycyrrhetinic acid and carbenoxolone (MacKenzieet al., J. Clin. Endocrinol. Metab., 1990, 70, 1637-1643; Stewart etal., Clin. Sci., 1990, 78, 49-54).

[0012] Disclosed and claimed in PCT publication WO 01/90093 is the useof 2-aminothiazole-derivatives as inhibitors of hydroxysteroid 11-betadehydrogenase 1 in the manufacture of medicaments for the prevention,management or treatment of diabetes, syndrome X, obesity, glaucoma,hyperlipidemia, hyperinsulinemia, osteoporosis, tuberculosis,depression, virus diseases and inflammatory disorders (Nilsson, 2001).

[0013] Disclosed and claimed in PCT publication WO 01/30383 is amedicament comprising hydroxysteroid 11-beta dehydrogenase 1 antisenseoligonucleotides combined with an antigen in order to improve andoptimize tolerance induction (Wilckens, 2001).

[0014] Antisense phosphorothioate oligonucleotides targeting the startcodon of human hydroxysteroid 11-beta dehydrogenase 1 have been employedin investigations of hydroxysteroid 11-beta dehydrogenase 1 activity andeffects on vascular contractile response and glucocorticoid metabolismin human vascular tissue (Hatakeyama et al., Front. Sci. Ser., 2000, 29,173-174; Souness et al., Steroids, 2002, 67, 195-201). Currently, thereare no known therapeutic agents that effectively inhibit the synthesisof hydroxysteroid 11-beta dehydrogenase 1. To date, investigativestrategies aimed at modulating hydroxysteroid 11-beta dehydrogenase 1expression have involved the use of small molecule inhibitors, geneknock-outs in mice and antisense oligonucleotides. Consequently, thereremains a long felt need for additional agents capable of effectivelyinhibiting hydroxysteroid 11-beta dehydrogenase 1 function.

[0015] Antisense technology is emerging as an effective means forreducing the expression of specific gene products and may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic, andresearch applications for the modulation of expression of hydroxysteroid11-beta dehydrogenase 1.

[0016] The present invention provides compositions and methods formodulating expression of hydroxysteroid 11-beta dehydrogenase 1.

SUMMARY OF THE INVENTION

[0017] The present invention is directed to compounds, particularlyantisense oligonucleotides, which are targeted to a nucleic acidencoding hydroxysteroid 11-beta dehydrogenase 1, and which modulate theexpression of hydroxysteroid 11-beta dehydrogenase 1. Pharmaceutical andother compositions comprising the compounds of the invention are alsoprovided. Further provided are methods of modulating the expression ofhydroxysteroid 11-beta dehydrogenase 1 in cells or tissues comprisingcontacting said cells or tissues with one or more of the antisensecompounds or compositions of the invention. Further provided are methodsof treating an animal, particularly a human, suspected of having orbeing prone to a disease or condition associated with expression ofhydroxysteroid 11-beta dehydrogenase 1 by administering atherapeutically or prophylactically effective amount of one or more ofthe antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding hydroxysteroid 11-beta dehydrogenase 1,ultimately modulating the amount of hydroxysteroid 11-beta dehydrogenase1 produced. This is accomplished by providing antisense compounds whichspecifically hybridize with one or more nucleic acids encodinghydroxysteroid 11-beta dehydrogenase 1. As used herein, the terms“target nucleic acid” and “nucleic acid encoding hydroxysteroid 11-betadehydrogenase 1” encompass DNA encoding hydroxysteroid 11-betadehydrogenase 1, RNA (including pre-mRNA and mRNA) transcribed from suchDNA, and also cDNA derived from such RNA. The specific hybridization ofan oligomeric compound with its target nucleic acid interferes with thenormal function of the nucleic acid. This modulation of function of atarget nucleic acid by compounds which specifically hybridize to it isgenerally referred to as “antisense”. The functions of DNA to beinterfered with include replication and transcription. The functions ofRNA to be interfered with include all vital functions such as, forexample, translocation of the RNA to the site of protein translation,translocation of the RNA to sites within the cell which are distant fromthe site of RNA synthesis, translation of protein from the RNA, splicingof the RNA to yield one or more mRNA species, and catalytic activitywhich may be engaged in or facilitated by the RNA. The overall effect ofsuch interference with target nucleic acid function is modulation of theexpression of hydroxysteroid 11-beta dehydrogenase 1. In the context ofthe present invention, “modulation” means either an increase(stimulation) or a decrease (inhibition) in the expression of a gene. Inthe context of the present invention, inhibition is the preferred formof modulation of gene expression and mRNA is a preferred target.

[0019] It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding hydroxysteroid 11-beta dehydrogenase 1. The targetingprocess also includes determination of a site or sites within this genefor the antisense interaction to occur such that the desired effect,e.g., detection or modulation of expression of the protein, will result.Within the context of the present invention, a preferred intragenic siteis the region encompassing the translation initiation or terminationcodon of the open reading frame (ORF) of the gene. Since, as is known inthe art, the translation initiation codon is typically 5′-AUG (intranscribed mRNA molecules; 5′-ATG in the corresponding DNA molecule),the translation initiation codon is also referred to as the “AUG codon,”the “start codon” or the “AUG start codon”. A minority of genes have atranslation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function invivo. Thus, the terms “translation initiation codon” and “start codon”can encompass many codon sequences, even though the initiator amino acidin each instance is typically methionine (in eukaryotes) orformylmethionine (in prokaryotes). It is also known in the art thateukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA molecule transcribedfrom a gene encoding hydroxysteroid 11-beta dehydrogenase 1, regardlessof the sequence(s) of such codons.

[0020] It is also known in the art that a translation termination codon(or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5¹-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

[0021] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Other target regions include the 5′untranslated region (5′UTR), known in the art to refer to the portion ofan mRNA in the 5′ direction from the translation initiation codon, andthus including nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5¹-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

[0022] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,intron-exon junctions, may also be preferred target regions, and areparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular mRNA spliceproduct is implicated in disease. Aberrant fusion junctions due torearrangements or deletions are also preferred targets. mRNA transcriptsproduced via the process of splicing of two (or more) mRNAs fromdifferent gene sources are known as “fusion transcripts”. It has alsobeen found that introns can also be effective, and therefore preferred,target regions for antisense compounds targeted, for example, to DNA orpre-mRNA.

[0023] It is also known in the art that alternative RNA transcripts canbe produced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic andextronic regions.

[0024] Upon excision of one or more exon or intron regions or portionsthereof during splicing, pre-mRNA variants produce smaller “mRNAvariants”. Consequently, mRNA variants are processed pre-mRNA variantsand each unique pre-mRNA variant must always produce a unique mRNAvariant as a result of splicing. These mRNA variants are also known as“alternative splice variants”. If no splicing of the pre-mRNA variantoccurs then the pre-mRNA variant is identical to the mRNA variant.

[0025] It is also known in the art that variants can be produced throughthe use of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites.

[0026] Once one or more target sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

[0027] In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

[0028] Antisense and other compounds of the invention which hybridize tothe target and inhibit expression of the target are identified throughexperimentation, and the sequences of these compounds are hereinbelowidentified as preferred embodiments of the invention. The target sitesto which these preferred sequences are complementary are hereinbelowreferred to as “active sites” and are therefore preferred sites fortargeting. Therefore another embodiment of the invention encompassescompounds which hybridize to these active sites, includingoligonucleotide probes and primers.

[0029] Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

[0030] For use in kits and diagnostics, the antisense compounds of thepresent invention, either alone or in combination with other antisensecompounds or therapeutics, can be used as tools in differential and/orcombinatorial analyses to elucidate expression patterns of a portion orthe entire complement of genes expressed within cells and tissues.

[0031] Expression patterns within cells or tissues treated with one ormore antisense compounds are compared to control cells or tissues nottreated with antisense compounds and the patterns produced are analyzedfor differential levels of gene expression as they pertain, for example,to disease association, signaling pathway, cellular localization,expression level, size, structure or function of the genes examined.These analyses can be performed on stimulated or unstimulated cells andin the presence or absence of other compounds which affect expressionpatterns.

[0032] Examples of methods of gene expression analysis known in the artinclude DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000,480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (reviewed in (To, Comb. Chem. High Throughput Screen, 2000, 3,235-41).

[0033] The specificity and sensitivity of antisense is also harnessed bythose of skill in the art for therapeutic uses. Antisenseoligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. Antisenseoligonucleotide drugs, including ribozymes, have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

[0034] In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics thereof. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

[0035] While antisense oligonucleotides are a preferred form ofantisense compound, the present invention comprehends other oligomericantisense compounds, including but not limited to oligonucleotidemimetics such as are described below. The antisense compounds inaccordance with this invention preferably comprise from about 8 to about80 nucleobases (i.e. from about 8 to about 80 linked nucleosides).Particularly preferred antisense compounds are antisenseoligonucleotides, even more preferably those comprising from about 12 toabout 30 nucleobases. Antisense compounds include ribozymes, externalguide sequence (EGS) oligonucleotides (oligozymes), and other shortcatalytic RNAs or catalytic oligonucleotides which hybridize to thetarget nucleic acid and modulate its expression.

[0036] As is known in the art, a nucleoside is a base-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. In addition, linearstructures may also have internal nucleobase complementarity and maytherefore fold in a manner as to produce a double stranded structure.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

[0037] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0038] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

[0039] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218;5,672,697 and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

[0040] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

[0041] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which Isherein incorporated by reference.

[0042] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0043] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0044] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O] _(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)CH₃)]₂, where n and m are from 1to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁D lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O—(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

[0045] Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

[0046] A further prefered modification includes Locked Nucleic Acids(LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbonatom of the sugar ring thereby forming a bicyclic sugar moiety. Thelinkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

[0047] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b] [1,4]benzoxazin-2(3H)-one),carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindolecytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modifiednucleobases may also include those in which the purine or pyrimidinebase is replaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsof the invention. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

[0048] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

[0049] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. The compounds of the inventioncan include conjugate groups covalently bound to functional groups suchas primary or secondary hydroxyl groups. Conjugate groups of theinvention include intercalators, reporter molecules, polyamines,polyamides, polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugates groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve oligomeruptake, enhance oligomer resistance to degradation, and/or strengthensequence-specific hybridization with RNA. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve oligomer uptake, distribution, metabolism orexcretion. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992 theentire disclosure of which is incorporated herein by reference.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937. Oligonucleotides of the invention mayalso be conjugated to active drug substances, for example, aspirin,warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

[0050] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference.

[0051] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,increased stability and/or increased binding affinity for the targetnucleic acid. An additional region of the oligonucleotide may serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof oligonucleotide inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as interferon-induced RNAseL whichcleaves both cellular and viral RNA. Consequently, comparable resultscan often be obtained with shorter oligonucleotides when chimericoligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

[0052] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety.

[0053] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0054] The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes, receptortargeted molecules, oral, rectal, topical or other formulations, forassisting in uptake, distribution and/or absorption. RepresentativeUnited States patents that teach the preparation of such uptake,distribution and/or absorption assisting formulations include, but arenot limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0055] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0056] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0057] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto.

[0058] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

[0059] For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts include but are not limited to (a) salts formed withcations such as sodium, potassium, ammonium, magnesium, calcium,polyamines such as spermine and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0060] The antisense compounds of the present invention can be utilizedfor diagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of hydroxysteroid 11-beta dehydrogenase 1 is treated byadministering antisense compounds in accordance with this invention. Thecompounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of an antisense compound to asuitable pharmaceutically acceptable diluent or carrier. Use of theantisense compounds and methods of the invention may also be usefulprophylactically, e.g., to prevent or delay infection, inflammation ortumor formation, for example.

[0061] The antisense compounds of the invention are useful for researchand diagnostics, because these compounds hybridize to nucleic acidsencoding hydroxysteroid 11-beta dehydrogenase 1, enabling sandwich andother assays to easily be constructed to exploit this fact.Hybridization of the antisense oligonucleotides of the invention with anucleic acid encoding hydroxysteroid 11-beta dehydrogenase 1 can bedetected by means known in the art. Such means may include conjugationof an enzyme to the oligonucleotide, radiolabelling of theoligonucleotide or any other suitable detection means. Kits using suchdetection means for detecting the level of hydroxysteroid 11-betadehydrogenase 1 in a sample may also be prepared.

[0062] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

[0063] Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful. Preferred topical formulationsinclude those in which the oligonucleotides of the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Preferred lipids and liposomes include neutral (e.g.dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). Oligonucleotides of the invention may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters include but are not limited arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999 which is incorporated herein byreference in its entirety.

[0064] Compositions and formulations for oral administration includepowders or granules, microparticulates, nanoparticulates, suspensions orsolutions in water or non-aqueous media, capsules, gel capsules,sachets, tablets or minitablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable. Preferred oralformulations are those in which oligonucleotides of the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Preferred surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Prefered bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate. Preferedfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also prefered are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly prefered combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents include poly-aminoacids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor oligonucleotides and their preparation are described in detail inU.S. application Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No.09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23,1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298(filed May 20, 1999) each of which is incorporated herein by referencein their entirety.

[0065] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0066] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

[0067] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0068] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0069] In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

[0070] Emulsions

[0071] The compositions of the present invention may be prepared andformulated as emulsions. Emulsions are typically heterogenous systems ofone liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter. (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising of two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be either water-in-oil (w/o) or of theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous provides an o/w/o emulsion.

[0072] Emulsions are characterized by little or no thermodynamicstability. Often, the dispersed or discontinuous phase of the emulsionis well dispersed into the external or continuous phase and maintainedin this form through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

[0073] Synthetic surfactants, also known as surface active agents, havefound wide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

[0074] Naturally occurring emulsifiers used in emulsion formulationsinclude lanolin, beeswax, phosphatides, lecithin and acacia. Absorptionbases possess hydrophilic properties such that they can soak up water toform w/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

[0075] A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0076] Hydrophilic colloids or hydrocolloids include naturally occurringgums and synthetic polymers such as polysaccharides (for example,acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, andtragacanth), cellulose derivatives (for example, carboxymethylcelluloseand carboxypropylcellulose), and synthetic polymers (for example,carbomers, cellulose ethers, and carboxyvinyl polymers). These disperseor swell in water to form colloidal solutions that stabilize emulsionsby forming strong interfacial films around the dispersed-phase dropletsand by increasing the viscosity of the external phase.

[0077] Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

[0078] The application of emulsion formulations via dermatological, oraland parenteral routes and methods for their manufacture have beenreviewed in the literature (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199). Emulsion formulations for oral deliveryhave been very widely used because of reasons of ease of formulation,efficacy from an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

[0079] In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0080] The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

[0081] Surfactants used in the preparation of microemulsions include,but are not limited to, ionic surfactants, non-ionic surfactants, Brij96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (Po500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

[0082] Microemulsions are particularly of interest from the standpointof drug solubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

[0083] Microemulsions of the present invention may also containadditional components and additives such as sorbitan monostearate (Grill3), Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

[0084] Liposomes

[0085] There are many organized surfactant structures besidesmicroemulsions that have been studied and used for the formulation ofdrugs. These include monolayers, micelles, bilayers and vesicles.Vesicles, such as liposomes, have attracted great interest because oftheir specificity and the duration of action they offer from thestandpoint of drug delivery. As used in the present invention, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in aspherical bilayer or bilayers.

[0086] Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

[0087] In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

[0088] Further advantages of liposomes include; liposomes obtained fromnatural phospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

[0089] Liposomes are useful for the transfer and delivery of activeingredients to the site of action. Because the liposomal membrane isstructurally similar to biological membranes, when liposomes are appliedto a tissue, the liposomes start to merge with the cellular membranes.As the merging of the liposome and cell progresses, the liposomalcontents are emptied into the cell where the active agent may act.

[0090] Liposomal formulations have been the focus of extensiveinvestigation as the mode of delivery for many drugs. There is growingevidence that for topical administration, liposomes present severaladvantages over other formulations. Such advantages include reducedside-effects related to high systemic absorption of the administereddrug, increased accumulation of the administered drug at the desiredtarget, and the ability to administer a wide variety of drugs, bothhydrophilic and hydrophobic, into the skin.

[0091] Several reports have detailed the ability of liposomes to deliveragents including high-molecular weight DNA into the skin. Compoundsincluding analgesics, antibodies, hormones and high-molecular weightDNAs have been administered to the skin. The majority of applicationsresulted in the targeting of the upper epidermis.

[0092] Liposomes fall into two broad classes. Cationic liposomes arepositively charged liposomes which interact with the negatively chargedDNA molecules to form a stable complex. The positively chargedDNA/liposome complex binds to the negatively charged cell surface and isinternalized in an endosome. Due to the acidic pH within the endosome,the liposomes are ruptured, releasing their contents into the cellcytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

[0093] Liposomes which are pH-sensitive or negatively-charged, entrapDNA rather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

[0094] One major type of liposomal composition includes phospholipidsother than naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0095] Several studies have assessed the topical delivery of liposomaldrug formulations to the skin. Application of liposomes containinginterferon to guinea pig skin resulted in a reduction of skin herpessores while delivery of interferon via other means (e.g. as a solutionor as an emulsion) were ineffective (Weiner et al., Journal of DrugTargeting, 1992, 2, 405-410). Further, an additional study tested theefficacy of interferon administered as part of a liposomal formulationto the administration of interferon using an aqueous system, andconcluded that the liposomal formulation was superior to aqueousadministration (du Plessis et al., Antiviral Research, 1992, 18,259-265).

[0096] Non-ionic liposomal systems have also been examined to determinetheir utility in the delivery of drugs to the skin, in particularsystems comprising non-ionic surfactant and cholesterol. Non-ionicliposomal formulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

[0097] Liposomes also include “sterically stabilized” liposomes, a termwhich, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposome(A) comprises one or more glycolipids, such as monosialogangliosideG_(M1), or (B) is derivatized with one or more hydrophilic polymers,such as a polyethylene glycol (PEG) moiety. While not wishing to bebound by any particular theory, it is thought in the art that, at leastfor sterically stabilized liposomes containing gangliosides,sphingomyelin, or PEG-derivatized lipids, the enhanced circulationhalf-life of these sterically stabilized liposomes derives from areduced uptake into cells of the reticuloendothelial system (RES) (Allenet al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993,53, 3765).

[0098] Various liposomes comprising one or more glycolipids are known inthe art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

[0099] Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

[0100] A limited number of liposomes comprising nucleic acids are knownin the art. WO 96/40062 to Thierry et al. discloses methods forencapsulating high molecular weight nucleic acids in liposomes. U.S.Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomesand asserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

[0101] Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

[0102] Surfactants find wide application in formulations such asemulsions (including microemulsions) and liposomes. The most common wayof classifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

[0103] If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

[0104] If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

[0105] If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

[0106] If the surfactant molecule has the ability to carry either apositive or negative charge, the surfactant is classified as amphoteric.Amphoteric surfactants include acrylic acid derivatives, substitutedalkylamides, N-alkylbetaines and phosphatides.

[0107] The use of surfactants in drug products, formulations and inemulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms,Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0108] Penetration Enhancers

[0109] In one embodiment, the present invention employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

[0110] Penetration enhancers may be classified as belonging to one offive broad categories, i.e., surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Eachof the above mentioned classes of penetration enhancers are describedbelow in greater detail.

[0111] Surfactants: In connection with the present invention,surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of oligonucleotidesthrough the mucosa is enhanced. In addition to bile salts and fattyacids, these penetration enhancers include, for example, sodium laurylsulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetylether) (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p.92); and perfluorochemical emulsions, such as FC-43.Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0112] Fatty acids: Various fatty acids and their derivatives which actas penetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0113] Bile salts: The physiological role of bile includes thefacilitation of dispersion and absorption of lipids and fat-solublevitamins (Brunton, Chapter 38 in: Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, NewYork, 1996, pp. 934-935). Various natural bile salts, and theirsynthetic derivatives, act as penetration enhancers. Thus the term “bilesalts” includes any of the naturally occurring components of bile aswell as any of their synthetic derivatives. The bile salts of theinvention include, for example, cholic acid (or its pharmaceuticallyacceptable sodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0114] Chelating Agents: Chelating agents, as used in connection withthe present invention, can be defined as compounds that remove metallicions from solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0115] Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

[0116] Agents that enhance uptake of oligonucleotides at the cellularlevel may also be added to the pharmaceutical and other compositions ofthe present invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

[0117] Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

[0118] Carriers

[0119] Certain compositions of the present invention also incorporatecarrier compounds in the formulation. As used herein, “carrier compound”or “carrier” can refer to a nucleic acid, or analog thereof, which isinert (i.e., does not possess biological activity per se) but isrecognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate oligonucleotide in hepatic tissue can bereduced when it is coadministered with polyinosinic acid, dextransulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense &Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0120] Excipients

[0121] In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

[0122] Pharmaceutically acceptable organic or inorganic excipientsuitable for non-parenteral administration which do not deleteriouslyreact with nucleic acids can also be used to formulate the compositionsof the present invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

[0123] Formulations for topical administration of nucleic acids mayinclude sterile and non-sterile aqueous solutions, non-aqueous solutionsin common solvents such as alcohols, or solutions of the nucleic acidsin liquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

[0124] Suitable pharmaceutically acceptable excipients include, but arenot limited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

[0125] Other Components

[0126] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

[0127] Aqueous suspensions may contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

[0128] Certain embodiments of the invention provide pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include but are notlimited to daunorubicin, daunomycin, dactinomycin, doxorubicin,epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

[0129] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

[0130] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in viva animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0131] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for OligonucleotideSynthesis Deoxy and 2′-alkoxy Amidites

[0132] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropylphosphoramidites were purchased from commercial sources (e.g. Chemgenes,Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxysubstituted nucleoside amidites are prepared as described in U.S. Pat.No. 5,506,351, herein incorporated by reference. For oligonucleotidessynthesized using 2′-alkoxy amidites, optimized synthesis cycles weredeveloped that incorporate multiple steps coupling longer wait timesrelative to standard synthesis cycles.

[0133] The following abbreviations are used in the text: thin layerchromatography (TLC), melting point (MP), high pressure liquidchromatography (HPLC), Nuclear Magnetic Resonance (NMR), argon (Ar),methanol (MeOH), dichloromethane (CH₂Cl₂), triethylamine (TEA), dimethylformamide (DMF), ethyl acetate (EtOAc), dimethyl sulfoxide (DMSO),tetrahydrofuran (THF).

[0134] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-dC)nucleotides were synthesized according to published methods (Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203) using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.) or prepared as follows:

[0135] Preparation of 5′-O-Dimethoxytrityl-thymidine Intermediate for5-methyl dC Amidite

[0136] To a 50 L glass reactor equipped with air stirrer and Ar gas linewas added thymidine (1.00 kg, 4.13 mol) in anhydrous pyridine (6 L) atambient temperature. Dimethoxytrityl (DMT) chloride (1.47 kg, 4.34 mol,1.05 eq) was added as a solid in four portions over 1 h. After 30 min,TLC indicated approx. 95% product, 2% thymidine, 5% DMT reagent andby-products and 2% 3′,5′-bis DMT product (Rf in EtOAc 0.45, 0.05, 0.98,0.95 respectively). Saturated sodium bicarbonate (4 L) and CH₂Cl₂ wereadded with stirring (pH of the aqueous layer 7.5). An additional 18 L ofwater was added, the mixture was stirred, the phases were separated, andthe organic layer was transferred to a second 50 L vessel. The aqueouslayer was extracted with additional CH₂Cl₂ (2×2 L). The combined organiclayer was washed with water (10 L) and then concentrated in a rotaryevaporator to approx. 3.6 kg total weight. This was redissolved inCH₂Cl₂ (3.5 L), added to the reactor followed by water (6 L) and hexanes(13 L). The mixture was vigorously stirred and seeded to give a finewhite suspended solid starting at the interface. After stirring for 1 h,the suspension was removed by suction through a ½″ diameter teflon tubeinto a 20 L suction flask, poured onto a 25 cm Coors Buchner funnel,washed with water (2×3 L) and a mixture of hexanes-CH₂Cl₂ (4:1, 2×3 L)and allowed to air dry overnight in pans (1″ deep). This was furtherdried in a vacuum oven (75° C., 0.1 mm Hg, 48 h) to a constant weight of2072 g (93%) of a white solid, (mp 122-124° C.). TLC indicated a tracecontamination of the bis DMT product. NMR spectroscopy also indicatedthat 1-2 mole percent pyridine and about 5 mole percent of hexanes wasstill present.

[0137] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidineIntermediate for 5-methyl-dC Amidite

[0138] To a 50 L Schott glass-lined steel reactor equipped with anelectric stirrer, reagent addition pump (connected to an additionfunnel), heating/cooling system, internal thermometer and an Ar gas linewas added 5′-O-dimethoxytrityl-thymidine (3.00 kg, 5.51 mol), anhydrousacetonitrile (25 L) and TEA (12.3 L, 88.4 mol, 16 eq). The mixture waschilled with stirring to −10° C. internal temperature (external −20°C.). Trimethylsilylchloride (2.1 L, 16.5 mol, 3.0 eq) was added over 30minutes while maintaining the internal temperature below −5° C.,followed by a wash of anhydrous acetonitrile (1 L). Note: the reactionis mildly exothermic and copious hydrochloric acid fumes form over thecourse of the addition. The reaction was allowed to warm to 0° C. andthe reaction progress was confirmed by TLC (EtOAc-hexanes 4:1; R_(f)0.43 to 0.84 of starting material and silyl product, respectively). Uponcompletion, triazole (3.05 kg, 44 mol, 8.0 eq) was added the reactionwas cooled to −20° C. internal temperature (external −30° C.).Phosphorous oxychloride (1035 mL, 11.1 mol, 2.01 eq) was added over 60min so as to maintain the temperature between −20° C. and −10° C. duringthe strongly exothermic process, followed by a wash of anhydrousacetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1h. TLC indicated a complete conversion to the triazole product (R_(f)0.83 to 0.34 with the product spot glowing in long wavelength UV light).The reaction mixture was a peach-colored thick suspension, which turneddarker red upon warming without apparent decomposition. The reaction wascooled to −15° C. internal temperature and water (5 L) was slowly addedat a rate to maintain the temperature below +10° C. in order to quenchthe reaction and to form a homogenous solution. (Caution: this reactionis initially very strongly exothermic). Approximately one-half of thereaction volume (22 L) was transferred by air pump to another vessel,diluted with EtOAc (12 L) and extracted with water (2×8 L). The combinedwater layers were back-extracted with EtOAc (6 L). The water layer wasdiscarded and the organic layers were concentrated in a 20 L rotaryevaporator to an oily foam. The foam was coevaporated with anhydrousacetonitrile (4 L) to remove EtOAc. (note: dioxane may be used insteadof anhydrous acetonitrile if dried to a hard foam). The second half ofthe reaction was treated in the same way. Each residue was dissolved indioxane (3 L) and concentrated ammonium hydroxide (750 mL) was added. Ahomogenous solution formed in a few minutes and the reaction was allowedto stand overnight (although the reaction is complete within 1 h).

[0139] TLC indicated a complete reaction (product R_(f) 0.35 inEtOAc-MeOH 4:1). The reaction solution was concentrated on a rotaryevaporator to a dense foam. Each foam was slowly redissolved in warmEtOAc (4 L; 50° C.), combined in a 50 L glass reactor vessel, andextracted with water (2×4L) to remove the triazole by-product. The waterwas back-extracted with EtOAc (2 L). The organic layers were combinedand concentrated to about 8 kg total weight, cooled to 0° C. and seededwith crystalline product. After 24 hours, the first crop was collectedon a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc (3×3L)until a white powder was left and then washed with ethyl ether (2×3L).The solid was put in pans (1″ deep) and allowed to air dry overnight.The filtrate was concentrated to an oil, then redissolved in EtOAc (2L), cooled and seeded as before. The second crop was collected andwashed as before (with proportional solvents) and the filtrate was firstextracted with water (2×1L) and then concentrated to an oil. The residuewas dissolved in EtOAc (1 L) and yielded a third crop which was treatedas above except that more washing was required to remove a yellow oilylayer.

[0140] After air-drying, the three crops were dried in a vacuum oven(50° C., 0.1 mm Hg, 24 h) to a constant weight (1750, 600 and 200 g,respectively) and combined to afford 2550 g (85%) of a white crystallineproduct (MP 215-217° C.) when TLC and NMR spectroscopy indicated purity.The mother liquor still contained mostly product (as determined by TLC)and a small amount of triazole (as determined by NMR spectroscopy), bisDMT product and unidentified minor impurities. If desired, the motherliquor can be purified by silica gel chromatography using a gradient ofMeOH (0-25%) in EtOAc to further increase the yield.

[0141] Preparation of5′-O-Dimethoxytrityl-2′-deoxy-N-4-benzoyl-5-methylcytidine PenultimateIntermediate for 5-methyl dC Amidite

[0142] Crystalline 5′-O-dimethoxytrityl-5-methyl-2′-deoxycytidine (2000g, 3.68 mol) was dissolved in anhydrous DMF (6.0 kg) at ambienttemperature in a 50 L glass reactor vessel equipped with an air stirrerand argon line. Benzoic anhydride (Chem Impex not Aldrich, 874 g, 3.86mol, 1.05 eq) was added and the reaction was stirred at ambienttemperature for 8 h. TLC (CH₂Cl₂-EtOAc; CH₂Cl₂-EtOAc 4:1; R_(f) 0.25)indicated approx. 92% complete reaction. An additional amount of benzoicanhydride (44 g, 0.19 mol) was added. After a total of 18 h, TLCindicated approx. 96% reaction completion. The solution was diluted withEtOAc (20 L), TEA (1020 mL, 7.36 mol, ca 2.0 eq) was added withstirring, and the mixture was extracted with water (15 L, then 2×10 L).The aqueous layer was removed (no back-extraction was needed) and theorganic layer was concentrated in 2×20 L rotary evaporator flasks untila foam began to form. The residues were coevaporated with acetonitrile(1.5 L each) and dried (0.1 mm Hg, 25° C., 24 h) to 2520 g of a densefoam. High pressure liquid chromatography (HPLC) revealed acontamination of 6.3% of N4, 3′-O-dibenzoyl product, but very littleother impurities.

[0143] THe product was purified by Biotage column chromatography (5 kgBiotage) prepared with 65:35:1 hexanes-EtOAc-TEA (4L). The crude product(800 g),dissolved in CH₂Cl₂ (2 L), was applied to the column. The columnwas washed with the 65:35:1 solvent mixture (20 kg), then 20:80:1solvent mixture (10 kg), then 99:1 EtOAc:TEA (17 kg). The fractionscontaining the product were collected, and any fractions containing theproduct and impurities were retained to be resubjected to columnchromatography. The column was re-equilibrated with the original 65:35:1solvent mixture (17 kg). A second batch of crude product (840 g) wasapplied to the column as before. The column was washed with thefollowing solvent gradients: 65:35:1 (9 kg), 55:45:1 (20 kg), 20:80:1(10 kg), and 99:1 EtOAc:TEA (15 kg). The column was reequilibrated asabove, and a third batch of the crude product (850 g) plus impurefractions recycled from the two previous columns (28 g) was purifiedfollowing the procedure for the second batch. The fractions containingpure product combined and concentrated on a 20L rotary evaporator,co-evaporated with acetontirile (3 L) and dried (0.1 mm Hg, 48 h, 25°C.) to a constant weight of 2023 g (85%) of white foam and 20 g ofslightly contaminated product from the third run. HPLC indicated apurity of 99.8% with the balance as the diBenzoyl product.

[0144][5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC Amidite)

[0145] 5′-O—(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidine(998 g, 1.5 mol) was dissolved in anhydrous DMF (2 L). The solution wasco-evaporated with toluene (300 ml) at 50° C. under reduced pressure,then cooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5g, 0.75 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (15 ml) was added and the mixture was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (2.5 L) and water (600 ml), and extracted with hexane(3×3 L). The mixture was diluted with water (1.2 L) and extracted with amixture of toluene (7.5 L) and hexane (6 L). The two layers wereseparated, the upper layer was washed with DMF-water (7:3 v/v, 3×2 L)and water (3×2 L), and the phases were separated. The organic layer wasdried (Na₂SO₄), filtered and rotary evaporated. The residue wasco-evaporated with acetonitrile (2×2 L) under reduced pressure and driedto a constant weight (25° C., 0.1 mm Hg, 40 h) to afford 1250 g anoff-white foam solid (96%).

[0146] 2′-Fluoro Amidites

[0147] 2′-Fluorodeoxyadenosine Amidites

[0148] 2′-fluoro oligonucleotides were synthesized as describedpreviously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] andU.S. Pat. No. 5,670,633, herein incorporated by reference. Thepreparation of 2′-fluoropyrimidines containing a 5-methyl substitutionare described in U.S. Pat. No. 5,861,493. Briefly, the protectednucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and whereby the 2′-alpha-fluoro atom is introduced bya S_(N)2-displacement of a 2′-beta-triflate group. ThusN6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected inmoderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N6-benzoyl groups was accomplished usingstandard methodologies to obtain the 5′-dimethoxytrityl-(DMT) and5′-DMT-3′-phosphoramidite intermediates.

[0149] 2′-Fluorodeoxyguanosine

[0150] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate isobutyryl-arabinofuranosylguanosine. Alternatively,isobutyryl-arabinofuranosylguanosine was prepared as described by Rosset al., (Nucleosides & Nucleosides, 16, 1645, 1997). Deprotection of theTPDS group was followed by protection of the hydroxyl group with THP togive isobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

[0151] 2′-Fluorouridine

[0152] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

[0153] 2′-Fluorodeoxycytidine

[0154] 2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0155] 2′-O-(2-Methoxyethyl) Modified Amidites

[0156] 2′-O-Methoxyethyl-substituted nucleoside amidites (otherwiseknown as MOE amidites) are prepared as follows, or alternatively, as perthe methods of Martin, P., (Helvetica Chimica Acta, 1995, 78, 486-504).

[0157] Preparation of 2′-O-(2-methoxyethyl)-5-methyluridine Intermediate

[0158] 2,2′-Anhydro-5-methyl-uridine (2000 g, 8.32 mol),tris(2-methoxyethyl)borate (2504 g, 10.60 mol), sodium bicarbonate (60g, 0.70 mol) and anhydrous 2-methoxyethanol (5 L) were combined in a 12L three necked flask and heated to 130° C. (internal temp) atatmospheric pressure, under an argon atmosphere with stirring for 21 h.TLC indicated a complete reaction. The solvent was removed under reducedpressure until a sticky gum formed (50-85° C. bath temp and 100-11 mmHg) and the residue was redissolved in water (3 L) and heated to boilingfor 30 min in order the hydrolyze the borate esters. The water wasremoved under reduced pressure until a foam began to form and then theprocess was repeated. HPLC indicated about 77% product, 15% dimer (5′ ofproduct attached to 2′ of starting material) and unknown derivatives,and the balance was a single unresolved early eluting peak.

[0159] The gum was redissolved in brine (3 L), and the flask was rinsedwith additional brine (3 L). The combined aqueous solutions wereextracted with chloroform (20 L) in a heavier-than continuous extractorfor 70 h. The chloroform layer was concentrated by rotary evaporation ina 20 L flask to a sticky foam (2400 g). This was coevaporated with MeOH(400 mL) and EtOAc (8 L) at 75° C. and 0.65 atm until the foam dissolvedat which point the vacuum was lowered to about 0.5 atm. After 2.5 L ofdistillate was collected a precipitate began to form and the flask wasremoved from the rotary evaporator and stirred until the suspensionreached ambient temperature. EtOAc (2 L) was added and the slurry wasfiltered on a 25 cm table top Buchner funnel and the product was washedwith EtOAc (3×2 L). The bright white solid was air dried in pans for 24h then further dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) toafford 1649 g of a white crystalline solid (mp 115.5-116.5° C.).

[0160] The brine layer in the 20 L continuous extractor was furtherextracted for 72 h with recycled chloroform. The chloroform wasconcentrated to 120 g of oil and this was combined with the motherliquor from the above filtration (225 g), dissolved in brine (250 mL)and extracted once with chloroform (250 mL). The brine solution wascontinuously extracted and the product was crystallized as describedabove to afford an additional 178 g of crystalline product containingabout 2% of thymine. The combined yield was 1827 g (69.4%). HPLCindicated about 99.5% purity with the balance being the dimer.

[0161] Preparation of 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridinePenultimate Intermediate

[0162] In a 50 L glass-lined steel reactor,2′-O-(2-methoxyethyl)-5-methyl-uridine (MOE-T, 1500 g, 4.738 mol),lutidine (1015 g, 9.476 mol) were dissolved in anhydrous acetonitrile(15 L). The solution was stirred rapidly and chilled to −10° C.(internal temperature). Dimethoxytriphenylmethyl chloride (1765.7 g,5.21 mol) was added as a solid in one portion. The reaction was allowedto warm to −2° C. over 1 h. (Note: The reaction was monitored closely byTLC (EtOAc) to determine when to stop the reaction so as to not generatethe undesired bis-DMT substituted side product). The reaction wasallowed to warm from −2 to 3° C. over 25 min. then quenched by addingMeOH (300 mL) followed after 10 min by toluene (16 L) and water (16 L).The solution was transferred to a clear 50 L vessel with a bottomoutlet, vigorously stirred for 1 minute, and the layers separated. Theaqueous layer was removed and the organic layer was washed successivelywith 10% aqueous citric acid (8 L) and water (12 L). The product wasthen extracted into the aqueous phase by washing the toluene solutionwith aqueous sodium hydroxide (0.5N, 16 L and 8 L). The combined aqueouslayer was overlayed with toluene (12 L) and solid citric acid (8 moles,1270 g) was added with vigorous stirring to lower the pH of the aqueouslayer to 5.5 and extract the product into the toluene. The organic layerwas washed with water (10 L) and TLC of the organic layer indicated atrace of DMT-O-Me, bis DMT and dimer DMT.

[0163] The toluene solution was applied to a silica gel column (6 Lsintered glass funnel containing approx. 2 kg of silica gel slurriedwith toluene (2 L) and TEA (25 mL)) and the fractions were eluted withtoluene (12 L) and EtOAc (3×4 L) using vacuum applied to a filter flaskplaced below the column. The first EtOAc fraction containing both thedesired product and impurities were resubjected to column chromatographyas above. The clean fractions were combined, rotary evaporated to afoam, coevaporated with acetonitrile (6 L) and dried in a vacuum oven(0.1 mm Hg, 40 h, 40° C.) to afford 2850 g of a white crisp foam. NMRspectroscopy indicated a 0.25 mole % remainder of acetonitrile(calculates to be approx. 47 g) to give a true dry weight of 2803 g(96%). HPLC indicated that the product was 99.41% pure, with theremainder being 0.06 DMT-O-Me, 0.10 unknown, 0.44 bis DMT, and nodetectable dimer DMT or 3′-O-DMT.

[0164] Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T Amidite)

[0165]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridine(1237 g, 2.0 mol) was dissolved in anhydrous DMF (2.5 L). The solutionwas co-evaporated with toluene (200 ml) at 50° C. under reducedpressure, then cooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (70 g,1.0 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (20 ml) was added and the solution was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (3.5 L) and water (600 ml) and extracted with hexane(3×3L). The mixture was diluted with water (1.6 L) and extracted withthe mixture of toluene (12 L) and hexanes (9 L). The upper layer waswashed with DMF-water (7:3 v/v, 3×3 L) and water (3×3 L). The organiclayer was dried (Na₂SO₄), filtered and evaporated. The residue wasco-evaporated with acetonitrile (2×2 L) under reduced pressure and driedin a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1526 g of anoff-white foamy solid (95%).

[0166] Preparation of5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine Intermediate

[0167] To a 50 L Schott glass-lined steel reactor equipped with anelectric stirrer, reagent addition pump (connected to an additionfunnel), heating/cooling system, internal thermometer and argon gas linewas added 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-uridine(2.616 kg, 4.23 mol, purified by base extraction only and no scrubcolumn), anhydrous acetonitrile (20 L), and TEA (9.5 L, 67.7 mol, 16eq). The mixture was chilled with stirring to −10° C. internaltemperature (external −20° C.). Trimethylsilylchloride (1.60 L, 12.7mol, 3.0 eq) was added over 30 min. while maintaining the internaltemperature below −5° C., followed by a wash of anhydrous acetonitrile(1 L). (Note: the reaction is mildly exothermic and copious hydrochloricacid fumes form over the course of the addition). The reaction wasallowed to warm to 0° C. and the reaction progress was confirmed by TLC(EtOAc, R_(f) 0.68 and 0.87 for starting material and silyl product,respectively). Upon completion, triazole (2.34 kg, 33.8 mol, 8.0 eq) wasadded the reaction was cooled to −20° C. internal temperature (external−30° C.). Phosphorous oxychloride (793 mL, 8.51 mol, 2.01 eq) was addedslowly over 60 min so as to maintain the temperature between −20° C. and−10° C. (note: strongly exothermic), followed by a wash of anhydrousacetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1h, at which point it was an off-white thick suspension. TLC indicated acomplete conversion to the triazole product (EtOAc, R_(f) 0.87 to 0.75with the product spot glowing in long wavelength UV light). The reactionwas cooled to −15° C. and water (5 L) was slowly added at a rate tomaintain the temperature below +10° C. in order to quench the reactionand to form a homogenous solution. (Caution: this reaction is initiallyvery strongly exothermic). Approximately one-half of the reaction volume(22 L) was transferred by air pump to another vessel, diluted with EtOAc(12 L) and extracted with water (2×8 L). The second half of the reactionwas treated in the same way. The combined aqueous layers wereback-extracted with EtOAc (8 L) The organic layers were combined andconcentrated in a 20 L rotary evaporator to an oily foam. The foam wascoevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note:dioxane may be used instead of anhydrous acetonitrile if dried to a hardfoam). The residue was dissolved in dioxane (2 L) and concentratedammonium hydroxide (750 mL) was added. A homogenous solution formed in afew minutes and the reaction was allowed to stand overnight

[0168] TLC indicated a complete reaction (CH₂Cl₂-acetone-MeOH, 20:5:3,R_(f) 0.51). The reaction solution was concentrated on a rotaryevaporator to a dense foam and slowly redissolved in warm CH₂Cl₂ (4 L,40° C.) and transferred to a 20 L glass extraction vessel equipped witha air-powered stirrer. The organic layer was extracted with water (2×6L) to remove the triazole by-product. (Note: In the first extraction anemulsion formed which took about 2 h to resolve). The water layer wasback-extracted with CH₂Cl₂ (2×2 L), which in turn was washed with water(3 L). The combined organic layer was concentrated in 2×20 L flasks to agum and then recrystallized from EtOAc seeded with crystalline product.After sitting overnight, the first crop was collected on a 25 cm CoorsBuchner funnel and washed repeatedly with EtOAc until a whitefree-flowing powder was left (about 3×3 L). The filtrate wasconcentrated to an oil recrystallized from EtOAc, and collected asabove. The solid was air-dried in pans for 48 h, then further dried in avacuum oven (50° C., 0.1 mm Hg, 17 h) to afford 2248 g of a brightwhite, dense solid (86%). An HPLC analysis indicated both crops to be99.4% pure and NMR spectroscopy indicated only a faint trace of EtOAcremained.

[0169] Preparation of5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N-4-benzoyl-5-methyl-cytidinePenultimate Intermediate:

[0170] Crystalline5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-cytidine (1000 g,1.62 mol) was suspended in anhydrous DMF (3 kg) at ambient temperatureand stirred under an Ar atmosphere. Benzoic anhydride (439.3 g, 1.94mol) was added in one portion. The solution clarified after 5 hours andwas stirred for 16 h. HPLC indicated 0.45% starting material remained(as well as 0.32% N4, 3′-O-bis Benzoyl). An additional amount of benzoicanhydride (6.0 g, 0.0265 mol) was added and after 17 h, HPLC indicatedno starting material was present. TEA (450 mL, 3.24 mol) and toluene (6L) were added with stirring for 1 minute. The solution was washed withwater (4×4 L), and brine (2×4 L). The organic layer was partiallyevaporated on a 20 L rotary evaporator to remove 4 L of toluene andtraces of water. HPLC indicated that the bis benzoyl side product waspresent as a 6% impurity. The residue was diluted with toluene (7 L) andanhydrous DMSO (200 mL, 2.82 mol) and sodium hydride (60% in oil, 70 g,1.75 mol) was added in one portion with stirring at ambient temperatureover 1 h. The reaction was quenched by slowly adding then washing withaqueous citric acid (10%, 100 mL over 10 min, then 2×4 L), followed byaqueous sodium bicarbonate (2%, 2 L), water (2×4 L) and brine (4 L). Theorganic layer was concentrated on a 20 L rotary evaporator to about 2 Ltotal volume. The residue was purified by silica gel columnchromatography (6 L Buchner funnel containing 1.5 kg of silica gelwetted with a solution of EtOAc-hexanes-TEA (70:29:1)). The product waseluted with the same solvent (30 L) followed by straight EtOAc (6 L).The fractions containing the product were combined, concentrated on arotary evaporator to a foam and then dried in a vacuum oven (50° C., 0.2mm Hg, 8 h) to afford 1155 g of a crisp, white foam (98%). HPLCindicated a purity of >99.7%.

[0171] Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C Amidite)

[0172]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidine(1082 g, 1.5 mol) was dissolved in anhydrous DMF (2 L) and co-evaporatedwith toluene (300 ml) at 50° C. under reduced pressure. The mixture wascooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5g, 0.75 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (30 ml) was added, and the mixture was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (1 L) and water (400 ml) and extracted with hexane (3×3L). The mixture was diluted with water (1.2 L) and extracted with amixture of toluene (9 L) and hexanes (6 L). The two layers wereseparated and the upper layer was washed with DMF-water (60:40 v/v, 3×3L) and water (3×2 L). The organic layer was dried (Na₂SO₄), filtered andevaporated. The residue was co-evaporated with acetonitrile (2×2 L)under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40h) to afford 1336 g of an off-white foam (97%).

[0173] Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A Amdite)

[0174]5′′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosine(purchased from Reliable Biopharmaceutical, St. Lois, Mo.), 1098 g, 1.5mol) was dissolved in anhydrous DMF (3 L) and co-evaporated with toluene(300 ml) at 50° C. The mixture was cooled to room temperature and2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) andtetrazole (78.8 g, 1.24 mol) were added. The mixture was shaken untilall tetrazole was dissolved, N-methylimidazole (30 ml) was added, andmixture was left at room temperature for 5 hours. TEA (300 ml) wasadded, the mixture was diluted with DMF (1 L) and water (400 ml) andextracted with hexanes (3×3 L). The mixture was diluted with water (1.4L) and extracted with the mixture of toluene (9 L) and hexanes (6 L).The two layers were separated and the upper layer was washed withDMF-water (60:40, v/v, 3×3 L) and water (3×2 L). The organic layer wasdried (Na₂SO₄), filtered and evaporated to a sticky foam. The residuewas co-evaporated with acetonitrile (2.5 L) under reduced pressure anddried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1350 g of anoff-white foam solid (96%).

[0175] Prepartion of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G Amidite)

[0176]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrlguanosine(purchased from Reliable Biopharmaceutical, St. Louis, Mo., 1426 g, 2.0mol) was dissolved in anhydrous DMF (2 L). The solution wasco-evaporated with toluene (200 ml) at 50° C., cooled to roomtemperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g,3.0 mol) and tetrazole (68 g, 0.97 mol) were added. The mixture wasshaken until all tetrazole was dissolved, N-methylimidazole (30 ml) wasadded, and the mixture was left at room temperature for 5 hours. TEA(300 ml) was added, the mixture was diluted with DMF (2 L) and water(600 ml) and extracted with hexanes (3×3 L). The mixture was dilutedwith water (2 L) and extracted with a mixture of toluene (10 L) andhexanes (5 L). The two layers were separated and the upper layer waswashed with DMF-water (60:40, v/v, 3×3 L). EtOAc (4 L) was added and thesolution was washed with water (3×4 L). The organic layer was dried(Na₂SO₄), filtered and evaporated to approx. 4 kg. Hexane (4 L) wasadded, the mixture was shaken for 10 min, and the supernatant liquid wasdecanted. The residue was co-evaporated with acetonitrile (2×2 L) underreduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) toafford 1660 g of an off-white foamy solid (91%).

[0177] 2′-O-(Aminooxyethyl) Nucleoside Amidites and2′-O-(dimethylaminooxyethyl) Nucleoside Amidites

[0178] 2′-(Dimethylaminooxyethoxy) Nucleoside Amidites

[0179] 2′-(Dimethylaminooxyethoxy) nucleoside amidites (also known inthe art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites) areprepared as described in the following paragraphs. Adenosine, cytidineand guanosine nucleoside amidites are prepared similarly to thethymidine (5-methyluridine) except the exocyclic amines are protectedwith a benzoyl moiety in the case of adenosine and cytidine and withisobutyryl in the case of guanosine.

[0180] 5′-O-tert-Butyldiphenylsilyl-0²-2′-anhydro-5-methyluridine

[0181] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054mmol) were dissolved in dry pyridine (500 ml) at ambient temperatureunder an argon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)was added in one portion. The reaction was stirred for 16 h at ambienttemperature. TLC (R_(f) 0.22, EtOAc) indicated a complete reaction. Thesolution was concentrated under reduced pressure to a thick oil. Thiswas partitioned between CH₂Cl₂ (1 L) and saturated sodium bicarbonate(2×1 L) and brine (1 L). The organic layer was dried over sodiumsulfate, filtered, and concentrated under reduced pressure to a thickoil. The oil was dissolved in a 1:1 mixture of EtOAc and ethyl ether(600 mL) and cooling the solution to −10° C. afforded a whitecrystalline solid which was collected by filtration, washed with ethylether (3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to afford 149 g ofwhite solid (74.8%). TLC and NMR spectroscopy were consistent with pureproduct.

[0182]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0183] In the fume hood, ethylene glycol (350 mL, excess) was addedcautiously with manual stirring to a 2 L stainless steel pressurereactor containing borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL).(Caution: evolves hydrogen gas).5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure <100 psig). The reaction vessel was cooled to ambienttemperature and opened. TLC (EtOAc, R_(f) 0.67 for desired product andR_(f) 0.82 for ara-T side product) indicated about 70% conversion to theproduct. The solution was concentrated under reduced pressure (10 to 1mm Hg) in a warm water bath (40-100° C.) with the more extremeconditions used to remove the ethylene glycol. (Alternatively, once theTHF has evaporated the solution can be diluted with water and theproduct extracted into EtOAc). The residue was purified by columnchromatography (2 kg silica gel, EtOAc-hexanes gradient 1:1 to 4:1). Theappropriate fractions were combined, evaporated and dried to afford 84 gof a white crisp foam (50%), contaminated starting material (17.4 g, 12%recovery) and pure reusable starting material (20 g, 13% recovery). TLCand NMR spectroscopy were consistent with 99% pure product.

[0184]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0185]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol) and dried over P₂O₅ underhigh vacuum for two days at 40° C. The reaction mixture was flushed withargon and dissolved in dry THF (369.8 mL, Aldrich, sure seal bottle).Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to thereaction mixture with the rate of addition maintained such that theresulting deep red coloration is just discharged before adding the nextdrop. The reaction mixture was stirred for 4 hrs., after which time TLC(EtOAc:hexane, 60:40) indicated that the reaction was complete. Thesolvent was evaporated in vacuuo and the residue purified by flashcolumn chromatography (eluted with 60:40 EtOAc:hexane), to yield 2′-O—([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%) upon rotary evaporation.

[0186]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0187]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate washed with ice coldCH₂Cl₂, and the combined organic phase was washed with water and brineand dried (anhydrous Na₂SO₄). The solution was filtered and evaporatedto afford 2′-O-(aminooxyethyl) thymidine, which was then dissolved inMeOH (67.5 mL). Formaldehyde (20% aqueous solution, w/w, 1.1 eq.) wasadded and the resulting mixture was stirred for 1 h. The solvent wasremoved under vacuum and the residue was purified by columnchromatography to yield5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%) upon rotaryevaporation.

[0188] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,Ndimethylaminooxyethyl]-5-methyluridine

[0189]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL) and cooled to 10° C.under inert atmosphere. Sodium cyanoborohydride (0.39 g, 6.13 mmol) wasadded and the reaction mixture was stirred. After 10 minutes thereaction was warmed to room temperature and stirred for 2 h. while theprogress of the reaction was monitored by TLC (5% MeOH in CH₂Cl₂).Aqueous NaHCO₃ solution (5%, 10 mL) was added and the product wasextracted with EtOAc (2×20 mL). The organic phase was dried overanhydrous Na₂SO₄, filtered, and evaporated to dryness. This entireprocedure was repeated with the resulting residue, with the exceptionthat formaldehyde (20% w/w, 30 mL, 3.37 mol) was added upon dissolutionof the residue in the PPTS/MeOH solution. After the extraction andevaporation, the residue was purified by flash column chromatography and(eluted with 5% MeOH in CH₂Cl₂) to afford5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%) upon rotary evaporation.

[0190] 2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0191] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolvedin dry THF and TEA (1.67 mL, 12 mmol, dry, stored over KOH) and added to5¹-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol). The reaction was stirred at room temperature for 24hrs and monitored by TLC (5% MeOH in CH₂Cl₂). The solvent was removedunder vacuum and the residue purified by flash column chromatography(eluted with 10% MeOH in CH₂Cl₂) to afford2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%) upon rotaryevaporation of the solvent.

[0192] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0193] 2′-O— (dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)was dried over P₂O₅ under high vacuum overnight at 40° C., co-evaporatedwith anhydrous pyridine (20 mL), and dissolved in pyridine (11 mL) underargon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol) and4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) were added to thepyridine solution and the reaction mixture was stirred at roomtemperature until all of the starting material had reacted. Pyridine wasremoved under vacuum and the residue was purified by columnchromatography (eluted with 10% MeOH in CH₂Cl₂ containing a few drops ofpyridine) to yield5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%)upon rotary evaporation.

[0194]5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-S-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0195] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g,1.67 mmol) was co-evaporated with toluene (20 mL), N,N-diisopropylaminetetrazonide (0.29 g, 1.67 mmol) was added and the mixture was dried overP₂O₅ under high vacuum overnight at 40° C. This was dissolved inanhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 h under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:EtOAc 1:1). The solvent was evaporated, thenthe residue was dissolved in EtOAc (70 mL) and washed with 5% aqueousNaHCO₃ (40 mL). The EtOAc layer was dried over anhydrous Na₂SO₄,filtered, and concentrated. The residue obtained was purified by columnchromatography (EtOAc as eluent) to afford5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%) upon rotary evaporation.

[0196] 2′-(Aminooxyethoxy) Nucleoside Amidites

[0197] 2′-(Aminooxyethoxy) nucleoside amidites (also known in the art as2′-O-(aminooxyethyl) nucleoside amidites) are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

[0198]N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0199] The 2′-O-aminooxyethyl guanosine analog may be obtained byselective 2′-O-alkylation of diaminopurine riboside. Multigramquantities of diaminopurine riboside may be purchased from Schering AG(Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside alongwith aminor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase.(McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.)Standard protection procedures should afford2′-O-(2-ethylacetyl)-51-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside may bephosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0200] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites

[0201] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known inthe art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂,or 2′-DMAEOE nucleoside amidites) are prepared as follows. Othernucleoside amidites are prepared similarly.

[0202] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine

[0203] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) wasslowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10mmol) with stirring in a 100 mL bomb. (Caution: Hydrogen gas evolves asthe solid dissolves). O²-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol),and sodium bicarbonate (2.5 mg) were added and the bomb was sealed,placed in an oil bath and heated to 155° C. for 26 h. then cooled toroom temperature. The crude solution was concentrated, the residue wasdiluted with water (200 mL) and extracted with hexanes (200 mL). Theproduct was extracted from the aqueous layer with EtOAc (3×200 mL) andthe combined organic layers were washed once with water, dried overanhydrous sodium sulfate, filtered and concentrated. The residue waspurified by silica gel column chromatography (eluted with 5:100:2MeOH/CH₂Cl₂/TEA) as the eluent. The appropriate fractions were combinedand evaporated to afford the product as a white solid.

[0204] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl Uridine

[0205] To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), was added TEA (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) and the reaction was stirred for 1 h. Thereaction mixture was poured into water (200 mL) and extracted withCH₂Cl₂ (2×200 mL). The combined CH₂Cl₂ layers were washed with saturatedNaHCO₃ solution, followed by saturated NaCl solution, dried overanhydrous sodium sulfate, filtered and evaporated. The residue waspurified by silica gel column chromatography (eluted with 5:100:1MeOH/CH₂Cl₂/TEA) to afford the product.

[0206]5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0207] Diisopropylaminotetrazolide (0.6 g) and2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) were addedto a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture was stirred overnight and the solventevaporated. The resulting residue was purified by silica gel columnchromatography with EtOAc as the eluent to afford the title compound.

Example 2

[0208] Oligonucleotide Synthesis

[0209] Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

[0210] Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄oAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

[0211] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0212] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0213] Phosphoramidite oligonucleotides are prepared as described inU.S. Patent, 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporatedby reference.

[0214] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0215] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0216] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0217] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

Example 3

[0218] Oligonucleoside Synthesis

[0219] Methylenemethylimino linked oligonucleosides, also identified asMMI linked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0220] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference.

[0221] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

[0222] PNA Synthesis

[0223] Peptide nucleic acids (PNAs) are prepared in accordance with anyof the various procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5

[0224] Synthesis of Chimeric Oligonucleotides

[0225] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[0226] [2′-O-Me]--[2′-deoxy]--[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

[0227] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligo-nucleotide segments are synthesizedusing an Applied Biosystems automated DNA synthesizer Model 394, asabove. Oligonucleotides are synthesized using the automated synthesizerand 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portionand 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′wings. The standard synthesis cycle is modified by incorporatingcoupling steps with increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) 12-16 hr at 55° C. The deprotected oligo isthen recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yeild and for purity by capillaryelectrophoresis and by mass spectrometry.

[0228] [2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O-(Methoxyethyl)]Chimeric Phosphorothioate Oligonucleotides

[0229] [2′-O-(2-methoxyethyl)]--[2′-deoxy]--[-2′-O-(methoxyethyl)]chimeric phosphorothioate oligonucleotides were prepared as per theprocedure above for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-0-(methoxyethyl) amidites for the 2′-O-methylamidites.

[0230] [2′-O-(2-Methoxyethyl)Phosphodiester]--[2′-deoxyPhosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[0231] [2′-O-(2-methoxyethyl phosphodiester]--[2′-deoxyphosphorothioate]--[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

[0232] Other chimeric oligonucleotides, chimeric oligonucleosides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

[0233] Oligonucleotide Isolation

[0234] After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes ethanol. Synthesized oligonucleotideswere analyzed by electrospray mass spectroscopy (molecular weightdetermination) and by capillary gel electrophoresis and judged to be atleast 70% full length material. The relative amounts of phosphorothioateand phosphodiester linkages obtained in synthesis was determined by theratio of correct molecular weight relative to the −16 amu product(+32+48). For some studies oligonucleotides were purified by HPLC, asdescribed by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171.Results obtained with HPLC-purified material were similar to thoseobtained with non-HPLC purified material.

Example 7

[0235] Oligonucleotide Synthesis—96 Well Plate Format

[0236] Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a 96 well format.Phosphodiester internucleotide linkages were afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages were generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

[0237] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8

[0238] Oligonucleotide Analysis—96 Well Plate Format

[0239] The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9

[0240] Cell Culture and Oligonucleotide Treatment

[0241] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,Ribonuclease protection assays, or RT-PCR.

[0242] T-24 Cells:

[0243] The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum ((Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

[0244] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0245] A549 Cells:

[0246] The human lung carcinoma cell line A549 was obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

[0247] NHDF Cells:

[0248] Human neonatal dermal fibroblast (NHDF) were obtained from theClonetics Corporation (Walkersville, Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corporation,Walkersville, MD) supplemented as recommended by the supplier. Cellswere maintained for up to 10 passages as recommended by the supplier.

[0249] HEK Cells:

[0250] Human embryonic keratinocytes (HEK) were obtained from theClonetics Corporation (Walkersville, Md.). HEKs were routinelymaintained in Keratinocyte Growth Medium (Clonetics Corporation,Walkersville, Md.) formulated as recommended by the supplier. Cells wereroutinely maintained for up to 10 passages as recommended by thesupplier.

[0251] THP-1 Cells:

[0252] The acute monocytic leukemia cell line, THP-1, was obtained fromthe American Type Culture Collection (ATCC) (Manassas, Va.). The cellsare routinely cultured at 37° C. in ATCC medium: RPMI 1640 medium with 2mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/Lglucose, 10 mM HEPES and 1.0 mM sodium pyruvate and supplemented with20% fetal bovine serum.

[0253] THP-1 cells are suspension cells and are therefore treated viaelectroporation. When the cell count reaches 2×10⁷ cells/mL, 90 ulvolumes of cell suspension are treated with 20 ul of 50 uMoligonucleotide (total concentration of 10 uM). This mixture is thentransferred to a 1 mm gap cuvette. Then, 75 V is applied to the cuvettefor 6 msec, after which, 800 ul growth media (RPMI) is added and mixedin the cuvette. 800 ul of this mixture is then transferred to a 24-wellplate already containing 1 mL of growth media (RPMI) in each well (togive a total volume of 1.9 mL). After incubation overnight at 5% CO₂,800 ul of the mixture is transferred in duplicate from the 24-well plateto a 96-well deep well block. After centrifugation for 10 minutes at2500 rpm, the media is decanted and cells are lysed in 150 ul of RLTbuffer (Quiagen, Valencia, Calif.). RNA harvested from these mixtures isused in the standard RT-PCR assay.

[0254] Treatment with Antisense Compounds:

[0255] When cells reached 70% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.After 4-7 hours of treatment, the medium was replaced with fresh medium.Cells were harvested 16-24 hours after oligonucleotide treatment.

[0256] The concentration of oligonucleotide used varies from cell lineto cell line. To determine the optimal oligonucleotide concentration fora particular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG,SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown inbold) with a phosphorothioate backbone which is targeted to human H-ras.For mouse or rat cells the positive control oligonucleotide is ISIS15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer(2′-O-methoxyethyls shown in bold) with a phosphorothioate backbonewhich is targeted to both mouse and rat c-raf. The concentration ofpositive control oligonucleotide that results in 80% inhibition ofc-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is thenutilized as the screening concentration for-new oligonucleotides insubsequent experiments for that cell line. If 80% inhibition is notachieved, the lowest concentration of positive control oligonucleotidethat results in 60% inhibition of H-ras or c-raf mRNA is then utilizedas the oligonucleotide screening concentration in subsequent experimentsfor that cell line. If 60% inhibition is not achieved, that particularcell line is deemed as unsuitable for oligonucleotide transfectionexperiments.

Example 10

[0257] Analysis of Oligonucleotide Inhibition of Hydroxysteroid 11-BetaDehydrogenase 1 Expression

[0258] Antisense modulation of hydroxysteroid 11-beta dehydrogenase 1expression can be assayed in a variety of ways known in the art. Forexample, hydroxysteroid 11-beta dehydrogenase 1 mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitativePCR is presently preferred. RNA analysis can be performed on totalcellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis ofthe present invention is the use of total cellular RNA as described inother examples herein. Methods of RNA isolation are taught in, forexample, Ausubel, F. M. et al., Current Protocols in Molecular Biology,Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993. Northern blot analysis is routine in the art and is taught in, forexample, Ausubel, F. M. et al., Current Protocols in Molecular Biology,Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-timequantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7700 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions.

[0259] Protein levels of hydroxysteroid 11-beta dehydrogenase 1 can bequantitated in a variety of ways well known in the art, such asimmunoprecipitation, Western blot analysis (immunoblotting), ELISA orfluorescence-activated cell sorting (FACS). Antibodies directed tohydroxysteroid 11-beta dehydrogenase 1 can be identified and obtainedfrom a variety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

[0260] Immunoprecipitation methods are standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons,Inc., 1998. Western blot (immunoblot) analysis is standard in the artand can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley& Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) arestandard in the art and can be found at, for example, Ausubel, F. M. etal., Current Protocols in Molecular Biology, Volume 2, pp.11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

Example 11

[0261] Poly(A)+ mRNA Isolation

[0262] Poly(A)+ mRNA was isolated according to Miura et al., Clin.Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolationare taught in, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993. Briefly, for cells grown on 96-well plates, growth medium wasremoved from the cells and each well was washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, theplate was gently agitated and then incubated at room temperature forfive minutes. 55 μL of lysate was transferred to Oligo d(T) coated96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60minutes at room temperature, washed 3 times with 200 μL of wash buffer(10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash,the plate was blotted on paper towels to remove excess wash buffer andthen air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH7.6), preheated to 70° C. was added to each well, the plate wasincubated on a 90° C. hot plate for 5 minutes, and the eluate was thentransferred to a fresh 96-well plate.

[0263] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

Example 12

[0264] Total RNA Isolation

[0265] Total RNA was isolated using an RNEASY 96™ kit and bufferspurchased from Qiagen Inc. (Valencia, Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium was removed from the cells and each wellwas washed with 200 μL cold PBS. 150 μL Buffer RLT was added to eachwell and the plate vigorously agitated for 20 seconds. 150 μL of 70%ethanol was then added to each well and the contents mixed by pipettingthree times up and down. The samples were then transferred to the RNEASY96™ well plate attached to a QIAVAC™ manifold fitted with a wastecollection tray and attached to a vacuum source. Vacuum was applied for1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and incubated for 15 minutes and the vacuum was again applied for1 minute. An additional 500 μL of Buffer RW1 was added to each well ofthe RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL ofBuffer RPE was then added to each well of the RNEASY 96™ plate and thevacuum applied for a period of 90 seconds. The Buffer RPE wash was thenrepeated and the vacuum was applied for an additional 3 minutes. Theplate was then removed from the QIAVAC™ manifold and blotted dry onpaper towels. The plate was then re-attached to the QIAVAC™ manifoldfitted with a collection tube rack containing 1.2 mL collection tubes.RNA was then eluted by pipetting 170 μL water into each well, incubating1 minute, and then applying the vacuum for 3 minutes.

[0266] The repetitive pipetting and elution steps may be automated usinga QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13

[0267] Real-Time Quantitative PCR Analysis of Hydroxysteroid 11-betaDehydrogenase 1 mRNA Levels

[0268] Quantitation of hydroxysteroid 11-beta dehydrogenase 1 mRNAlevels was determined by real-time quantitative PCR using the ABI PRISM™7700 Sequence Detection System (PE-Applied Biosystems, Foster City,Calif.) according to manufacturer's instructions. This is a closed-tube,non-gel-based, fluorescence detection system which allowshigh-throughput quantitation of polymerase chain reaction (PCR) productsin real-time. As opposed to standard PCR, in which amplificationproducts are quantitated after the PCR is completed, products inreal-time quantitative PCR are quantitated as they accumulate. This isaccomplished by including in the PCR reaction an oligonucleotide probethat anneals specifically between the forward and reverse PCR primers,and contains two fluorescent dyes. A reporter dye (e.g., FAM, obtainedfrom either Operon Technologies Inc., Alameda, CA or Integrated DNATechnologies Inc., Coralville, Iowa) is attached to the 5′ end of theprobe and a quencher dye (e.g., TAMRA, obtained from either OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 3′ end of the probe. When the probeand dyes are intact, reporter dye emission is quenched by the proximityof the 3′ quencher dye. During amplification, annealing of the probe tothe target sequence creates a substrate that can be cleaved by the5′-exonuclease activity of Taq polymerase. During the extension phase ofthe PCR amplification cycle, cleavage of the probe by Taq polymerasereleases the reporter dye from the remainder of the probe (and hencefrom the quencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™7700 Sequence Detection System. In each assay, a series of parallelreactions containing serial dilutions of mRNA from untreated controlsamples generates a standard curve that is used to quantitate thepercent inhibition after antisense oligonucleotide treatment of testsamples.

[0269] Prior to quantitative PCR analysis, primer-probe sets specific tothe target gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

[0270] PCR reagents were obtained from Invitrogen, Carlsbad, Calif.RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCRbuffer (—MgCl₂), 6.6 mM MgCl₂, 375 μM each of DATP, dCTP, dCTP and dGTP,375 nM each of forward primer and reverse primer, 125 nM of probe, 4Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reversetranscriptase, and 2.5×ROX dye) to 96 well plates containing 30 μL totalRNA solution. The RT reaction was carried out by incubation for 30minutes at 48° C. Following a 10 minute incubation at 95° C. to activatethe PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carriedout: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5minutes (annealing/extension).

[0271] Gene target quantities obtained by real time RT-PCR arenormalized using either the expression level of GAPDH, a gene whoseexpression is constant, or by quantifying total RNA using RiboGreen™(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen™RNA quantification reagent from Molecular Probes. Methods of RNAquantification by RiboGreenTM are taught in Jones, L. J., et al,Analytical Biochemistry, 1998, 265, 368-374.

[0272] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipettedinto a 96-well plate containing 30 μL purified, cellular RNA. The plateis read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at480 nm and emission at 520 nm.

[0273] Probes and primers to human hydroxysteroid 11-beta dehydrogenase1 were designed to hybridize to a human hydroxysteroid 11-betadehydrogenase 1 sequence, using published sequence information (GenBankaccession number NM_(—)005525.1, incorporated herein as SEQ ID NO:3).For human hydroxysteroid 11-beta dehydrogenase 1 the PCR primers were:forward primer: GTTTCTGGGATAGTCCATATGCAA (SEQ ID NO: 4) reverse primer:AGCTCCCCCTTTGATGATCTC (SEQ ID NO: 5) and the PCR probe was:FAM-CAGCTCCAAAGGAGGAATGTGCCCT-TAMRA (SEQ ID NO: 6) where FAM (PE-AppliedBiosystems, Foster City, Calif.) is the fluorescent reporter dye) andTAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.For human GAPDH the PCR primers were:

[0274] forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:7)

[0275] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:8) and the PCRprobe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 9) where JOE(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is thequencher dye.

[0276] Probes and primers to mouse hydroxysteroid 11-beta dehydrogenase1 were designed to hybridize to a mouse hydroxysteroid 11-betadehydrogenase 1 sequence, using published sequence information (GenBankaccession number X83202.1, incorporated herein as SEQ ID NO:10). Formouse hydroxysteroid 11-beta dehydrogenase 1 the PCR primers were:forward primer: GGCGGACTGGACATGCTT (SEQ ID NO:11) reverse primer:GAGTGGATGTCGTCATGGAAGA (SEQ ID NO: 12) and the PCR probe was:FAM-TTCTAAACCACATCACTCAGACCTCGCTGTC-TAMRA (SEQ ID NO: 13) where FAM(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is thequencher dye. For mouse GAPDH the PCR primers were:

[0277] forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO:14)

[0278] reverse primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO:15) and the PCRprobe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 16)where JOE (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye.

Example 14

[0279] Northern Blot Analysis of Hydroxysteroid 11-beta Dehydrogenase 1mRNA Levels

[0280] Eighteen hours after antisense treatment, cell monolayers werewashed twice with cold PBS and lysed in 1 mL RNAZOLTM (TEL-TEST “B”Inc., Friendswood, Tex.). Total RNA was prepared followingmanufacturer's recommended protocols. Twenty micrograms of total RNA wasfractionated by electrophoresis through 1.2% agarose gels containing1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon,Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes(Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillarytransfer using a Northern/Southern Transfer buffer system (TEL-TEST “B”Inc., Friendswood, Tex.). RNA transfer was confirmed by UVvisualization. Membranes were fixed by UV cross-linking using aSTRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.)and then probed using QUICKHYB™ hybridization solution (Stratagene, LaJolla, Calif.) using manufacturer's recommendations for stringentconditions.

[0281] To detect human hydroxysteroid 11-beta dehydrogenase 1, a humanhydroxysteroid 11-beta dehydrogenase 1 specific probe was prepared byPCR using the forward primer GTTTCTGGGATAGTCCATATGCAA (SEQ ID NO: 4) andthe reverse primer AGCTCCCCCTTTGATGATCTC (SEQ ID NO: 5). To normalizefor variations in loading and transfer efficiency membranes werestripped and probed for human glyceraldehyde-3-phosphate dehydrogenase(GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0282] To detect mouse hydroxysteroid 11-beta dehydrogenase 1, a mousehydroxysteroid 11-beta dehydrogenase 1 specific probe was prepared byPCR using the forward primer GGCGGACTGGACATGCTT (SEQ ID NO: 11) and thereverse primer GAGTGGATGTCGTCATGGAAGA (SEQ ID NO: 12). To normalize forvariations in loading and transfer efficiency membranes were strippedand probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH)RNA (Clontech, Palo Alto, Calif.).

[0283] Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15

[0284] Antisense Inhibition of Human Hydroxysteroid 11-betaDehydrogenase 1 Expression by Chimeric Phosphorothioate Oligonucleotideshaving 2′-MOE Wings and a Deoxy Gap

[0285] In accordance with the present invention, a series ofoligonucleotides were designed to target different regions of the humanhydroxysteroid 11-beta dehydrogenase 1 RNA, using published sequences(GenBank accession number NM_(—)005525.1, incorporated herein as SEQ IDNO: 3). The oligonucleotides are shown in Table 1. “Target site”indicates the first (5′-most) nucleotide number on the particular targetsequence to which the oligonucleotide binds. All compounds in Table 1are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length,composed of a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human hydroxysteroid 11-beta dehydrogenase 1 mRNA levelsby quantitative real-time PCR as described in other examples herein.Data are averages from two experiments. If present, “N.D.” indicates “nodata”. TABLE 1 Inhibition of human hydroxysteroid 11-beta dehydrogenase1 mRNA levels by chimeric phosphorothioate oligonucleotides having2′-MOE wings and a deoxy gap TARGET SEQ ID TARGET SEQ ID ISIS # REGIONNO SITE SEQUENCE % INHIB NO 145986 coding 3 141 agaatagtagtagtaggcca 4517 145990 coding 3 194 gtgacaatcactttctttcc 83 18 146007 coding 3 555ctgcttcagcatgggcaagg 39 19 146008 coding 3 561 attgctctgcttcagcatgg 5120 146020 coding 3 737 tctgtgtctatgaggccaag 45 21 146021 coding 3 743gctgtttctgtgtctatgag 36 22 146024 coding 3 818 cctttgatgatctccagggc 9023 242358 5′ UTR 3 5 ctaagcaggcagcagcctct 71 24 242361 5′ UTR 3 25acagagctttctacaacctc 63 25 242364 start 3 86 ataaaagccatccgacaggg 49 26codon 242367 coding 3 175 cttggagcatctctggtctg 60 27 242370 coding 3 300taccaccttctgtagagttt 64 28 242372 coding 3 321 tccaagctccaggcagtggg 4929 242374 coding 3 350 gtgccagcaatgtagtgtgc 76 30 242376 coding 3 382caaattgctctgcgaaggtc 77 31 242378 coding 3 411 tcctcccatgagctttcctg 5732 242380 coding 3 428 agaatgagcatgtctagtcc 58 33 242382 coding 3 484gcacatggtgaatatcatca 89 34 242384 coding 3 520 ccacgtaactgaggaagttg 4935 242386 coding 3 573 aacaatgcttccattgctct 40 36 242388 coding 3 597tttcccagccagagaggaga 44 37 242390 coding 3 647 tccaaagcaaacttgcttgc 4338 242392 coding 3 714 acagagagtgattgatacat 44 39 242394 coding 3 771atggactatcccagaaactg 86 40 242396 coding 3 790 cctttggagctgcttgcata 9741 242398 coding 3 822 tccccctttgatgatctcca 70 42 242400 coding 3 838cttcttggcgcagagctccc 58 43 242402 coding 3 872 agagtggtccagagtgagct 5444 242404 coding 3 881 ctgatcagaagagtggtcca 57 45 242406 coding 3 918tgagtagagaaattccagga 58 46 242408 3′ UTR 3 979 agcatgcccagccctcaggg 6447 242410 3′ UTR 3 992 cccaaaatccctcagcatgc 87 48 242412 3′ UTR 3 1043gggaagatgtcttcatagat 57 49 242414 3′ UTR 3 1080 gtgtgacccatgacttgcat 7750 242416 3′ UTR 3 1092 tccatttgtcaggtgtgacc 89 51 242418 3′ UTR 3 1125catttccattttgcaaatgt 65 52 242422 3′ UTR 3 1188 cctatgtttactaacaattt 5153 242424 3′ UTR 3 1307 tagctaccagttatgaattt 42 54 242426 3′ UTR 3 1334agaaccatcctgaataagct 46 55 242428 3′ UTR 3 1353 tgtacagtttatggtttaaa 1956

[0286] As shown in Table 1, SEQ ID NOs 18, 20, 23, 24, 25, 27, 28, 30,31, 32, 33, 34, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 and53 demonstrated at least 51% inhibition of human hydroxysteroid 11-betadehydrogenase 1 expression in this assay and are therefore preferred.The target sites to which these preferred sequences are complementaryare herein referred to as “active sites” and are therefore preferredsites for targeting by compounds of the present invention.

Example 16

[0287] Antisense Inhibition of Mouse Hydroxysteroid 11-betaDehydrogenase 1 Expression by Chimeric Phosphorothioate Oligonucleotideshaving 2′-MOE Wings and a Deoxy Gap.

[0288] In accordance with the present invention, a second series ofoligonucleotides were designed to target different regions of the mousehydroxysteroid 11-beta dehydrogenase 1 RNA, using a published sequence(GenBank accession number X83202.1, incorporated herein as SEQ ID NO:10). The oligonucleotides are shown in Table 2. “Target site” indicatesthe first (5′-most) nucleotide number on the particular target sequenceto which the oligonucleotide binds. All compounds in Table 2 arechimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composedof a central “gap” region consisting of ten 2′-deoxynucleotides, whichis flanked on both sides (5′ and 3′ directions) by five-nucleotide“wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides.The internucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on mousehydroxysteroid 11-beta dehydrogenase 1 mRNA levels by quantitativereal-time PCR as described in other examples herein. Data are averagesfrom two experiments. If present, “N.D.” indicates “no data”. TABLE 2Inhibition of mouse hydroxysteroid 11-beta dehydrogenase 1 mRNA levelsby chimeric phosphorothioate oligonucleotides having 2′-MOE wings and adeoxy gap TARGET SEQ ID TARGET SEQ ID ISIS # REGION NO SITE SEQUENCE %INHIB NO 145979 5′ UTR 10 2 ctctatccttctgtctcatc 0 57 145980 5′ UTR 1081 gaggcttcagataagtgcgg 0 58 145981 5′ UTR 10 101 ggacctggctcaggcccctt19 59 145982 start 10 110 atcaaacagggacctggctc 38 60 codon 145983 start10 119 ataactgccatcaaacaggg 25 61 codon 145984 coding 10 150gagcaccaggatcgggagga 21 62 145985 coding 10 157 ccagggagagcaccaggatc 863 145986 coding 10 174 agaatagtagtagtaggcca 0 17 145987 coding 10 183ttcatttgtagaatagtagt 0 64 145988 coding 10 193 gtctgaactcttcatttgta 0 65145989 coding 10 203 agcatttctggtctgaactc 21 66 145990 coding 10 227gtgacaatcactttctttcc 30 18 145991 coding 10 234 ggccccagtgacaatcactt 067 145992 coding 10 240 tttgctggccccagtgacaa 28 68 145993 coding 10 252tcttccaatccctttgctgg 37 69 145994 coding 10 270 cagatgatatgccatttctc 570 145995 coding 10 280 ccatttttgacagatgatat 0 71 145996 coding 10 290acatgggctcccatttttga 11 72 145997 coding 10 300 agtcaataccacatgggctc 1773 145998 coding 10 350 agttcaaggcagcgagacac 19 74 145999 coding 10 360ggctgctccgagttcaaggc 31 75 146000 coding 10 370 agtgagcagaggctgctccg 2776 146001 coding 10 417 aataaattgctccgcaaatg 12 77 146002 coding 10 440cccatgagctttcccgcctt 0 78 146003 coding 10 483 cgaggtctgagtgatgtggt 9479 146004 coding 10 514 cagagtggatgtcgtcatgg 46 80 146005 coding 10 536ttgacctccatgactcttcg 21 81 146006 coding 10 544 tgaggaagttgacctccatg 3982 146007 coding 10 588 ctgcttcagcatgggcaagg 29 19 146008 coding 10 594attgctctgcttcagcatgg 32 20 146009 coding 10 600 gctgccattgctctgcttca 983 146010 coding 10 605 gcaatgctgccattgctctg 26 84 146011 coding 10 611atgacggcaatgctgccatt 16 85 146012 coding 10 645 cataggctgggtcattttcc 1186 146013 coding 10 672 aaacttgcttgcagagtagg 25 87 146014 coding 10 682catccagagcaaacttgctt 49 88 146015 coding 10 692 gaaaagaacccatccagagc 089 146016 coding 10 702 tctaatggtggaaaagaacc 3 90 146017 coding 10 724ccttggttatgtagagttct 17 91 146018 coding 10 737 atggacacgttgaccttggt 5292 146019 coding 10 747 acagagagtgatggacacgt 13 93 146020 coding 10 770tctgtgtctatgaggccaag 0 21 146021 coding 10 776 gctgtttctgtgtctatgag 7122 146022 coding 10 803 tcaattatcccagagatttc 17 94 146023 coding 10 843gatctccagggcgcactcct 35 95 146024 coding 10 851 cctttgatgatctccagggc 123 146025 coding 10 857 gctgtgcctttgatgatctc 43 96 146026 coding 10 867tttgcgtagagctgtgcctt 36 97 146027 coding 10 894 aggcaatttgtcatagtaca 1698 146028 coding 10 913 caagcaggattggagtcaaa 22 99 146029 coding 10 935atgatcttccttcctgggtt 27 100 146030 coding 10 941 aattccatgatcttccttcc 36101 146031 coding 10 972 catgtccttattataatatc 11 102 146032 stop 10 996caggagttcctagttactta 15 103 codon 146033 3′ UTR 10 1018tctaagaccactcaccaggg 81 104 146034 3′ UTR 10 1047 gggcttactgaagtatgagg10 105 146035 3′ UTR 10 1073 tctctggaaagatacttttg 66 106 146036 3′ UTR10 1082 atttgtgtatctctggaaag 40 107 146037 3′ UTR 10 1092gtaccccaaaatttgtgtat 64 108 146038 3′ UTR 10 1107 ttctcatgatgaggtgtacc84 109 146039 3′ UTR 10 1119 tgttgcaagaatttctcatg 61 110 146040 3′ UTR10 1129 actgtgcaagtgttgcaaga 45 111 146041 3′ UTR 10 1138tacattttcactgtgcaagt 51 112 146042 3′ UTR 10 1154 tgacatttattacaattaca40 113 146043 3′ UTR 10 1160 ggtttgtgacatttattaca 71 114 146044 3′ UTR10 1166 caaagtggtttgtgacattt 10 115 146045 3′ UTR 10 1187tcaagttcacaactgcaggc 71 116 146046 3′ UTR 10 1198 catagttacaatcaagttca28 117 146047 3′ UTR 10 1220 acaaccactatgtgtttata 26 118 146048 3′ UTR10 1227 agccgatacaaccactatgt 74 119 146049 3′ UTR 10 1248gtttcattcagtgtgaggta 44 120 146050 3′ UTR 10 1296 gaagttgatattacctttat44 121 146051 3′ UTR 10 1301 tttacgaagttgatattacc 0 122

[0289] As shown in Table 2, SEQ ID NOs 22, 79, 80, 88, 92, 96, 104, 106,107, 108, 109, 110, 111, 112, 113, 114, 116, 119, 120 and 121demonstrated at least 40% inhibition of mouse hydroxysteroid 11-betadehydrogenase 1 expression in this experiment and are thereforepreferred. The target sites to which these preferred sequences arecomplementary are herein referred to as “active sites” and are thereforepreferred sites for targeting by compounds of the present invention.

Example 17

[0290] Western Blot Analysis of Hydroxysteroid 11-beta Dehydrogenase

[0291] 1 Protein Levels

[0292] Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to hydroxysteroid11-beta dehydrogenase 1 is used, with a radiolabelled or fluorescentlylabeled secondary antibody directed against the primary antibodyspecies. Bands are visualized using a PHOSPHORIMAGE™ (MolecularDynamics, Sunnyvale Calif.).

1 122 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence AntisenseOligonucleotide 2 atgcattctg cccccaagga 20 3 1375 DNA Homo sapiens CDS(95)...(973) 3 attcagaggc tgctgcctgc ttaggaggtt gtagaaagct ctgtaggttctctctgtgtg 60 tcctacagga gtcttcaggc cagctccctg tcgg atg gct ttt atg aaaaaa tat 115 Met Ala Phe Met Lys Lys Tyr 1 5 ctc ctc ccc att ctg ggg ctcttc atg gcc tac tac tac tat tct gca 163 Leu Leu Pro Ile Leu Gly Leu PheMet Ala Tyr Tyr Tyr Tyr Ser Ala 10 15 20 aac gag gaa ttc aga cca gag atgctc caa gga aag aaa gtg att gtc 211 Asn Glu Glu Phe Arg Pro Glu Met LeuGln Gly Lys Lys Val Ile Val 25 30 35 aca ggg gcc agc aaa ggg atc gga agagag atg gct tat cat ctg gcg 259 Thr Gly Ala Ser Lys Gly Ile Gly Arg GluMet Ala Tyr His Leu Ala 40 45 50 55 aag atg gga gcc cat gtg gtg gtg acagcg agg tca aaa gaa act cta 307 Lys Met Gly Ala His Val Val Val Thr AlaArg Ser Lys Glu Thr Leu 60 65 70 cag aag gtg gta tcc cac tgc ctg gag cttgga gca gcc tca gca cac 355 Gln Lys Val Val Ser His Cys Leu Glu Leu GlyAla Ala Ser Ala His 75 80 85 tac att gct ggc acc atg gaa gac atg acc ttcgca gag caa ttt gtt 403 Tyr Ile Ala Gly Thr Met Glu Asp Met Thr Phe AlaGlu Gln Phe Val 90 95 100 gcc caa gca gga aag ctc atg gga gga cta gacatg ctc att ctc aac 451 Ala Gln Ala Gly Lys Leu Met Gly Gly Leu Asp MetLeu Ile Leu Asn 105 110 115 cac atc acc aac act tct ttg aat ctt ttt catgat gat att cac cat 499 His Ile Thr Asn Thr Ser Leu Asn Leu Phe His AspAsp Ile His His 120 125 130 135 gtg cgc aaa agc atg gaa gtc aac ttc ctcagt tac gtg gtc ctg act 547 Val Arg Lys Ser Met Glu Val Asn Phe Leu SerTyr Val Val Leu Thr 140 145 150 gta gct gcc ttg ccc atg ctg aag cag agcaat gga agc att gtt gtc 595 Val Ala Ala Leu Pro Met Leu Lys Gln Ser AsnGly Ser Ile Val Val 155 160 165 gtc tcc tct ctg gct ggg aaa gtg gct tatcca atg gtt gct gcc tat 643 Val Ser Ser Leu Ala Gly Lys Val Ala Tyr ProMet Val Ala Ala Tyr 170 175 180 tct gca agc aag ttt gct ttg gat ggg ttcttc tcc tcc atc aga aag 691 Ser Ala Ser Lys Phe Ala Leu Asp Gly Phe PheSer Ser Ile Arg Lys 185 190 195 gaa tat tca gtg tcc agg gtc aat gta tcaatc act ctc tgt gtt ctt 739 Glu Tyr Ser Val Ser Arg Val Asn Val Ser IleThr Leu Cys Val Leu 200 205 210 215 ggc ctc ata gac aca gaa aca gcc atgaag gca gtt tct ggg ata gtc 787 Gly Leu Ile Asp Thr Glu Thr Ala Met LysAla Val Ser Gly Ile Val 220 225 230 cat atg caa gca gct cca aag gag gaatgt gcc ctg gag atc atc aaa 835 His Met Gln Ala Ala Pro Lys Glu Glu CysAla Leu Glu Ile Ile Lys 235 240 245 ggg gga gct ctg cgc caa gaa gaa gtgtat tat gac agc tca ctc tgg 883 Gly Gly Ala Leu Arg Gln Glu Glu Val TyrTyr Asp Ser Ser Leu Trp 250 255 260 acc act ctt ctg atc aga aat cca tgcagg aag atc ctg gaa ttt ctc 931 Thr Thr Leu Leu Ile Arg Asn Pro Cys ArgLys Ile Leu Glu Phe Leu 265 270 275 tac tca acg agc tat aat atg gac agattc ata aac aag tag 973 Tyr Ser Thr Ser Tyr Asn Met Asp Arg Phe Ile AsnLys * 280 285 290 gaactccctg agggctgggc atgctgaggg attttgggac tgttctgtctcatgtttatc 1033 tgagctctta tctatgaaga catcttccca gagtgtcccc agagacatgcaagtcatggg 1093 tcacacctga caaatggaag gagttcctct aacatttgca aaatggaaatgtaataataa 1153 tgaatgtcat gcaccgctgc agccagcagt tgtaaaattg ttagtaaacataggtataat 1213 taccagatag ttatattaaa tttatatctt atatataata atatgtgatgattaatacaa 1273 tattaattat aataaaggtc acataaactt tataaattca taactggtagctataacttg 1333 agcttattca ggatggttct ttaaaccata aactgtacaa tg 1375 4 24DNA Artificial Sequence PCR Primer 4 gtttctggga tagtccatat gcaa 24 5 21DNA Artificial Sequence PCR Primer 5 agctccccct ttgatgatct c 21 6 25 DNAArtificial Sequence PCR Probe 6 cagctccaaa ggaggaatgt gccct 25 7 19 DNAArtificial Sequence PCR Primer 7 gaaggtgaag gtcggagtc 19 8 20 DNAArtificial Sequence PCR Primer 8 gaagatggtg atgggatttc 20 9 20 DNAArtificial Sequence PCR Probe 9 caagcttccc gttctcagcc 20 10 1350 DNA Musmusculus CDS (128)...(1006) 10 ggatgagaca gaaggataga gaggaggagagagagagaga gaagagaagc aaccagaaat 60 aggcagccaa taaaaaggag ccgcacttatctgaagcctc aaggggcctg agccaggtcc 120 ctgtttg atg gca gtt atg aaa aat tacctc ctc ccg atc ctg gtg ctc 169 Met Ala Val Met Lys Asn Tyr Leu Leu ProIle Leu Val Leu 1 5 10 tcc ctg gcc tac tac tac tat tct aca aat gaa gagttc aga cca gaa 217 Ser Leu Ala Tyr Tyr Tyr Tyr Ser Thr Asn Glu Glu PheArg Pro Glu 15 20 25 30 atg ctc cag gga aag aaa gtg att gtc act ggg gccagc aaa ggg att 265 Met Leu Gln Gly Lys Lys Val Ile Val Thr Gly Ala SerLys Gly Ile 35 40 45 gga aga gaa atg gca tat cat ctg tca aaa atg gga gcccat gtg gta 313 Gly Arg Glu Met Ala Tyr His Leu Ser Lys Met Gly Ala HisVal Val 50 55 60 ttg act gcc agg tcg gag gaa ggt ctc cag aag gta gtg tctcgc tgc 361 Leu Thr Ala Arg Ser Glu Glu Gly Leu Gln Lys Val Val Ser ArgCys 65 70 75 ctt gaa ctc gga gca gcc tct gct cac tac att gct ggc act atggaa 409 Leu Glu Leu Gly Ala Ala Ser Ala His Tyr Ile Ala Gly Thr Met Glu80 85 90 gac atg aca ttt gcg gag caa ttt att gtc aag gcg gga aag ctc atg457 Asp Met Thr Phe Ala Glu Gln Phe Ile Val Lys Ala Gly Lys Leu Met 95100 105 110 ggc gga ctg gac atg ctt att cta aac cac atc act cag acc tcgctg 505 Gly Gly Leu Asp Met Leu Ile Leu Asn His Ile Thr Gln Thr Ser Leu115 120 125 tct ctc ttc cat gac gac atc cac tct gtg cga aga gtc atg gaggtc 553 Ser Leu Phe His Asp Asp Ile His Ser Val Arg Arg Val Met Glu Val130 135 140 aac ttc ctc agc tac gtg gtc atg agc aca gcc gcc ttg ccc atgctg 601 Asn Phe Leu Ser Tyr Val Val Met Ser Thr Ala Ala Leu Pro Met Leu145 150 155 aag cag agc aat ggc agc att gcc gtc atc tcc tcc ttg gct gggaaa 649 Lys Gln Ser Asn Gly Ser Ile Ala Val Ile Ser Ser Leu Ala Gly Lys160 165 170 atg acc cag cct atg att gct ccc tac tct gca agc aag ttt gctctg 697 Met Thr Gln Pro Met Ile Ala Pro Tyr Ser Ala Ser Lys Phe Ala Leu175 180 185 190 gat ggg ttc ttt tcc acc att aga aca gaa ctc tac ata accaag gtc 745 Asp Gly Phe Phe Ser Thr Ile Arg Thr Glu Leu Tyr Ile Thr LysVal 195 200 205 aac gtg tcc atc act ctc tgt gtc ctt ggc ctc ata gac acagaa aca 793 Asn Val Ser Ile Thr Leu Cys Val Leu Gly Leu Ile Asp Thr GluThr 210 215 220 gct atg aag gaa atc tct ggg ata att gac gcc cta gct tctccc aag 841 Ala Met Lys Glu Ile Ser Gly Ile Ile Asp Ala Leu Ala Ser ProLys 225 230 235 gag gag tgc gcc ctg gag atc atc aaa ggc aca gct cta cgcaaa agc 889 Glu Glu Cys Ala Leu Glu Ile Ile Lys Gly Thr Ala Leu Arg LysSer 240 245 250 gag gtg tac tat gac aaa ttg cct ttg act cca atc ctg cttggg aac 937 Glu Val Tyr Tyr Asp Lys Leu Pro Leu Thr Pro Ile Leu Leu GlyAsn 255 260 265 270 cca gga agg aag atc atg gaa ttt ttt tca tta cga tattat aat aag 985 Pro Gly Arg Lys Ile Met Glu Phe Phe Ser Leu Arg Tyr TyrAsn Lys 275 280 285 gac atg ttt gta agt aac tag gaactcctga gccctggtgagtggtcttag 1036 Asp Met Phe Val Ser Asn * 290 aacagtcctg cctcatacttcagtaagccc tacccacaaa agtatctttc cagagataca 1096 caaattttgg ggtacacctcatcatgagaa attcttgcaa cacttgcaca gtgaaaatgt 1156 aattgtaata aatgtcacaaaccactttgg gcctgcagtt gtgaacttga ttgtaactat 1216 ggatataaac acatagtggttgtatcggct ttacctcaca ctgaatgaaa caatgataac 1276 taatgtaaca ttaaatataataaaggtaat atcaacttcg taaatgcaaa aaaaaaaaaa 1336 aaaaaaaaaa aaaa 1350 1118 DNA Artificial Sequence PCR Primer 11 ggcggactgg acatgctt 18 12 22DNA Artificial Sequence PCR Primer 12 gagtggatgt cgtcatggaa ga 22 13 31DNA Artificial Sequence PCR Probe 13 ttctaaacca catcactcag acctcgctgt c31 14 20 DNA Artificial Sequence PCR Primer 14 ggcaaattca acggcacagt 2015 20 DNA Artificial Sequence PCR Primer 15 gggtctcgct cctggaagat 20 1627 DNA Artificial Sequence PCR Probe 16 aaggccgaga atgggaagct tgtcatc 2717 20 DNA Artificial Sequence Antisense Oligonucleotide 17 agaatagtagtagtaggcca 20 18 20 DNA Artificial Sequence Antisense Oligonucleotide 18gtgacaatca ctttctttcc 20 19 20 DNA Artificial Sequence AntisenseOligonucleotide 19 ctgcttcagc atgggcaagg 20 20 20 DNA ArtificialSequence Antisense Oligonucleotide 20 attgctctgc ttcagcatgg 20 21 20 DNAArtificial Sequence Antisense Oligonucleotide 21 tctgtgtcta tgaggccaag20 22 20 DNA Artificial Sequence Antisense Oligonucleotide 22 gctgtttctgtgtctatgag 20 23 20 DNA Artificial Sequence Antisense Oligonucleotide 23cctttgatga tctccagggc 20 24 20 DNA Artificial Sequence AntisenseOligonucleotide 24 ctaagcaggc agcagcctct 20 25 20 DNA ArtificialSequence Antisense Oligonucleotide 25 acagagcttt ctacaacctc 20 26 20 DNAArtificial Sequence Antisense Oligonucleotide 26 ataaaagcca tccgacaggg20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27 cttggagcatctctggtctg 20 28 20 DNA Artificial Sequence Antisense Oligonucleotide 28taccaccttc tgtagagttt 20 29 20 DNA Artificial Sequence AntisenseOligonucleotide 29 tccaagctcc aggcagtggg 20 30 20 DNA ArtificialSequence Antisense Oligonucleotide 30 gtgccagcaa tgtagtgtgc 20 31 20 DNAArtificial Sequence Antisense Oligonucleotide 31 caaattgctc tgcgaaggtc20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32 tcctcccatgagctttcctg 20 33 20 DNA Artificial Sequence Antisense Oligonucleotide 33agaatgagca tgtctagtcc 20 34 20 DNA Artificial Sequence AntisenseOligonucleotide 34 gcacatggtg aatatcatca 20 35 20 DNA ArtificialSequence Antisense Oligonucleotide 35 ccacgtaact gaggaagttg 20 36 20 DNAArtificial Sequence Antisense Oligonucleotide 36 aacaatgctt ccattgctct20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37 tttcccagccagagaggaga 20 38 20 DNA Artificial Sequence Antisense Oligonucleotide 38tccaaagcaa acttgcttgc 20 39 20 DNA Artificial Sequence AntisenseOligonucleotide 39 acagagagtg attgatacat 20 40 20 DNA ArtificialSequence Antisense Oligonucleotide 40 atggactatc ccagaaactg 20 41 20 DNAArtificial Sequence Antisense Oligonucleotide 41 cctttggagc tgcttgcata20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42 tccccctttgatgatctcca 20 43 20 DNA Artificial Sequence Antisense Oligonucleotide 43cttcttggcg cagagctccc 20 44 20 DNA Artificial Sequence AntisenseOligonucleotide 44 agagtggtcc agagtgagct 20 45 20 DNA ArtificialSequence Antisense Oligonucleotide 45 ctgatcagaa gagtggtcca 20 46 20 DNAArtificial Sequence Antisense Oligonucleotide 46 tgagtagaga aattccagga20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47 agcatgcccagccctcaggg 20 48 20 DNA Artificial Sequence Antisense Oligonucleotide 48cccaaaatcc ctcagcatgc 20 49 20 DNA Artificial Sequence AntisenseOligonucleotide 49 gggaagatgt cttcatagat 20 50 20 DNA ArtificialSequence Antisense Oligonucleotide 50 gtgtgaccca tgacttgcat 20 51 20 DNAArtificial Sequence Antisense Oligonucleotide 51 tccatttgtc aggtgtgacc20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52 catttccattttgcaaatgt 20 53 20 DNA Artificial Sequence Antisense Oligonucleotide 53cctatgttta ctaacaattt 20 54 20 DNA Artificial Sequence AntisenseOligonucleotide 54 tagctaccag ttatgaattt 20 55 20 DNA ArtificialSequence Antisense Oligonucleotide 55 agaaccatcc tgaataagct 20 56 20 DNAArtificial Sequence Antisense Oligonucleotide 56 tgtacagttt atggtttaaa20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57 ctctatccttctgtctcatc 20 58 20 DNA Artificial Sequence Antisense Oligonucleotide 58gaggcttcag ataagtgcgg 20 59 20 DNA Artificial Sequence AntisenseOligonucleotide 59 ggacctggct caggcccctt 20 60 20 DNA ArtificialSequence Antisense Oligonucleotide 60 atcaaacagg gacctggctc 20 61 20 DNAArtificial Sequence Antisense Oligonucleotide 61 ataactgcca tcaaacaggg20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62 gagcaccaggatcgggagga 20 63 20 DNA Artificial Sequence Antisense Oligonucleotide 63ccagggagag caccaggatc 20 64 20 DNA Artificial Sequence AntisenseOligonucleotide 64 ttcatttgta gaatagtagt 20 65 20 DNA ArtificialSequence Antisense Oligonucleotide 65 gtctgaactc ttcatttgta 20 66 20 DNAArtificial Sequence Antisense Oligonucleotide 66 agcatttctg gtctgaactc20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67 ggccccagtgacaatcactt 20 68 20 DNA Artificial Sequence Antisense Oligonucleotide 68tttgctggcc ccagtgacaa 20 69 20 DNA Artificial Sequence AntisenseOligonucleotide 69 tcttccaatc cctttgctgg 20 70 20 DNA ArtificialSequence Antisense Oligonucleotide 70 cagatgatat gccatttctc 20 71 20 DNAArtificial Sequence Antisense Oligonucleotide 71 ccatttttga cagatgatat20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72 acatgggctcccatttttga 20 73 20 DNA Artificial Sequence Antisense Oligonucleotide 73agtcaatacc acatgggctc 20 74 20 DNA Artificial Sequence AntisenseOligonucleotide 74 agttcaaggc agcgagacac 20 75 20 DNA ArtificialSequence Antisense Oligonucleotide 75 ggctgctccg agttcaaggc 20 76 20 DNAArtificial Sequence Antisense Oligonucleotide 76 agtgagcaga ggctgctccg20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77 aataaattgctccgcaaatg 20 78 20 DNA Artificial Sequence Antisense Oligonucleotide 78cccatgagct ttcccgcctt 20 79 20 DNA Artificial Sequence AntisenseOligonucleotide 79 cgaggtctga gtgatgtggt 20 80 20 DNA ArtificialSequence Antisense Oligonucleotide 80 cagagtggat gtcgtcatgg 20 81 20 DNAArtificial Sequence Antisense Oligonucleotide 81 ttgacctcca tgactcttcg20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82 tgaggaagttgacctccatg 20 83 20 DNA Artificial Sequence Antisense Oligonucleotide 83gctgccattg ctctgcttca 20 84 20 DNA Artificial Sequence AntisenseOligonucleotide 84 gcaatgctgc cattgctctg 20 85 20 DNA ArtificialSequence Antisense Oligonucleotide 85 atgacggcaa tgctgccatt 20 86 20 DNAArtificial Sequence Antisense Oligonucleotide 86 cataggctgg gtcattttcc20 87 20 DNA Artificial Sequence Antisense Oligonucleotide 87 aaacttgcttgcagagtagg 20 88 20 DNA Artificial Sequence Antisense Oligonucleotide 88catccagagc aaacttgctt 20 89 20 DNA Artificial Sequence AntisenseOligonucleotide 89 gaaaagaacc catccagagc 20 90 20 DNA ArtificialSequence Antisense Oligonucleotide 90 tctaatggtg gaaaagaacc 20 91 20 DNAArtificial Sequence Antisense Oligonucleotide 91 ccttggttat gtagagttct20 92 20 DNA Artificial Sequence Antisense Oligonucleotide 92 atggacacgttgaccttggt 20 93 20 DNA Artificial Sequence Antisense Oligonucleotide 93acagagagtg atggacacgt 20 94 20 DNA Artificial Sequence AntisenseOligonucleotide 94 tcaattatcc cagagatttc 20 95 20 DNA ArtificialSequence Antisense Oligonucleotide 95 gatctccagg gcgcactcct 20 96 20 DNAArtificial Sequence Antisense Oligonucleotide 96 gctgtgcctt tgatgatctc20 97 20 DNA Artificial Sequence Antisense Oligonucleotide 97 tttgcgtagagctgtgcctt 20 98 20 DNA Artificial Sequence Antisense Oligonucleotide 98aggcaatttg tcatagtaca 20 99 20 DNA Artificial Sequence AntisenseOligonucleotide 99 caagcaggat tggagtcaaa 20 100 20 DNA ArtificialSequence Antisense Oligonucleotide 100 atgatcttcc ttcctgggtt 20 101 20DNA Artificial Sequence Antisense Oligonucleotide 101 aattccatgatcttccttcc 20 102 20 DNA Artificial Sequence Antisense Oligonucleotide102 catgtcctta ttataatatc 20 103 20 DNA Artificial Sequence AntisenseOligonucleotide 103 caggagttcc tagttactta 20 104 20 DNA ArtificialSequence Antisense Oligonucleotide 104 tctaagacca ctcaccaggg 20 105 20DNA Artificial Sequence Antisense Oligonucleotide 105 gggcttactgaagtatgagg 20 106 20 DNA Artificial Sequence Antisense Oligonucleotide106 tctctggaaa gatacttttg 20 107 20 DNA Artificial Sequence AntisenseOligonucleotide 107 atttgtgtat ctctggaaag 20 108 20 DNA ArtificialSequence Antisense Oligonucleotide 108 gtaccccaaa atttgtgtat 20 109 20DNA Artificial Sequence Antisense Oligonucleotide 109 ttctcatgatgaggtgtacc 20 110 20 DNA Artificial Sequence Antisense Oligonucleotide110 tgttgcaaga atttctcatg 20 111 20 DNA Artificial Sequence AntisenseOligonucleotide 111 actgtgcaag tgttgcaaga 20 112 20 DNA ArtificialSequence Antisense Oligonucleotide 112 tacattttca ctgtgcaagt 20 113 20DNA Artificial Sequence Antisense Oligonucleotide 113 tgacatttattacaattaca 20 114 20 DNA Artificial Sequence Antisense Oligonucleotide114 ggtttgtgac atttattaca 20 115 20 DNA Artificial Sequence AntisenseOligonucleotide 115 caaagtggtt tgtgacattt 20 116 20 DNA ArtificialSequence Antisense Oligonucleotide 116 tcaagttcac aactgcaggc 20 117 20DNA Artificial Sequence Antisense Oligonucleotide 117 catagttacaatcaagttca 20 118 20 DNA Artificial Sequence Antisense Oligonucleotide118 acaaccacta tgtgtttata 20 119 20 DNA Artificial Sequence AntisenseOligonucleotide 119 agccgataca accactatgt 20 120 20 DNA ArtificialSequence Antisense Oligonucleotide 120 gtttcattca gtgtgaggta 20 121 20DNA Artificial Sequence Antisense Oligonucleotide 121 gaagttgatattacctttat 20 122 20 DNA Artificial Sequence Antisense Oligonucleotide122 tttacgaagt tgatattacc 20

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targetedto a nucleic acid molecule encoding hydroxysteroid 11-beta dehydrogenase1, wherein said compound specifically hybridizes with said nucleic acidmolecule encoding hydroxysteroid 11-beta dehydrogenase 1 and inhibitsthe expression of hydroxysteroid 11-beta dehydrogenase
 1. 2. Thecompound of claim 1 which is an antisense oligonucleotide.
 3. Thecompound of claim 2 wherein the antisense oligonucleotide has a sequencecomprising SEQ ID NO: 20, 21, 22, 25, 26, 27, 28, 30, 31, 32, 33, 34,35, 39, 40, 41, 42, 45, 46, 47, 49, 50, 51, 52, 53, 55, 57, 58, 59, 60,61, 64, 65, 66, 67, 68, 73, 76, 78, 80, 82, 84, 86, 88, 89, 91, 92, 94,97, 23, 24, 36, 37, 101, 102, 103, 105, 106, 107, 108, 109, 110, 111,112, 113, 115, 117, 118, 119, 120, 121, 122, 125, 129, 130, 131, 132,133, 134, 135, 139, 143, 145 or
 155. 4. The compound of claim 2 whereinthe antisense oligonucleotide comprises at least one modifiedinternucleoside linkage.
 5. The compound of claim 4 wherein the modifiedinternucleoside linkage is a phosphorothioate linkage.
 6. The compoundof claim 2 wherein the antisense oligonucleotide comprises at least onemodified sugar moiety.
 7. The compound of claim 6 wherein the modifiedsugar moiety is a 2′-O-methoxyethyl sugar moiety.
 8. The compound ofclaim 2 wherein the antisense oligonucleotide comprises at least onemodified nucleobase.
 9. The compound of claim 8 wherein the modifiednucleobase is a 5-methylcytosine.
 10. The compound of claim 2 whereinthe antisense oligonucleotide is a chimeric oligonucleotide.
 11. Acompound 8 to 80 nucleobases in length which specifically hybridizeswith at least an 8-nucleobase portion of an active site on a nucleicacid molecule encoding hydroxysteroid 11-beta dehydrogenase
 1. 12. Acomposition comprising the compound of claim 1 and a pharmaceuticallyacceptable carrier or diluent.
 13. The composition of claim 12 furthercomprising a colloidal dispersion system.
 14. The composition of claim12 wherein the compound is an antisense oligonucleotide.
 15. A method ofinhibiting the expression of hydroxysteroid 11-beta dehydrogenase 1 incells or tissues comprising contacting said cells or tissues with thecompound of claim 1 so that expression of hydroxysteroid 11-betadehydrogenase 1 is inhibited.
 16. A method of treating an animal havinga disease or condition associated with hydroxysteroid 11-betadehydrogenase 1 comprising administering to said animal atherapeutically or prophylactically effective amount of the compound ofclaim 1 so that expression of hydroxysteroid 11-beta dehydrogenase 1 isinhibited.
 17. The method of claim 16 wherein the disease or conditionis a metabolic disorder.
 18. The method of claim 17 wherein themetabolic disorder is selected from the group consisting of obesity,diabetes, atherosclerosis and hyperlipidemia.
 19. The method of claim 16wherein the disease or condition is osteoporosis.
 20. The method ofclaim 16 wherein the disease or condition is depression.